Antibodies that bind human C6 and uses thereof

ABSTRACT

Isolated monoclonal antibodies that bind to human complement component C6 and related antibody-based compositions and molecules are disclosed. Also disclosed are therapeutic methods for using the antibodies.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/IB2015/002504, filed on Dec. 18, 2015, which claims priority to, andthe benefit of, U.S. Provisional Application No. 62/094,649, filed onDec. 19, 2014. The contents of the aforementioned applications arehereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 16, 2017, isnamed RGJ-005PCCN_SL.txt and is 52,064 bytes in size.

BACKGROUND OF THE INVENTION

The complement system is part of the innate immune system that functionsto aid, or “complement”, antibodies and phagocytic cells in theclearance of pathogens from an organism. Upon activation of the system,a catalytic set of reactions and interactions occur, resulting in thetargeting of the activating cell, organism or particle for destruction.The complement system comprises a set of over 30 plasma and membraneproteins that act together in a regulated cascade system to attackextracellular forms of pathogens (e.g., bacterium). The complementsystem includes two distinct enzymatic activation cascades, theclassical and alternative pathways, which converge in a common terminalnon-enzymatic pathway known as the membrane attack pathway.

The first enzymatically-activated cascade, known as the classicalpathway, comprises several components, C1, C4, C2, C3 and C5 (listed byorder in the pathway). Initiation of the classical pathway of thecomplement system occurs following binding and activation of the firstcomplement component (C1) by both immune and non-immune activators. C1comprises a calcium-dependent complex of components C1q, C1r and C1s,and is activated through binding of the C1q component. C1q contains sixidentical subunits and each subunit comprises three chains (the A, B andC chains). Each chain has a globular head region that is connected to acollagen-like tail. Binding and activation of C1q by antigen-antibodycomplexes occurs through the C1q head group region. Numerousnon-antibody C1 q activators, including proteins, lipids and nucleicacids, bind and activate C1q through a distinct site on thecollagen-like stalk region. The C1qrs complex then catalyzes theactivation of complement components C4 and C2, forming the C4b2a complexwhich functions as a C3 convertase.

The second enzymatically-activated cascade, known as the alternativepathway, is a rapid, antibody-independent route for complement systemactivation and amplification. The alternative pathway comprises severalcomponents, C3, factor B, and factor D (listed by order in the pathway).Activation of the alternative pathway occurs when C3b, a proteolyticallycleaved form of C3, is bound to an activating surface agent such as abacterium. Factor B is then bound to C3b, and cleaved by factor D toyield the active enzyme, Ba. The enzyme Ba then cleaves more C3 togenerate more C3b, producing extensive deposition of C3b-Ba complexes onthe activating surface.

Thus, both the classical and alternate complement pathways produce C3convertases that split factor C3 into C3a and C3b. At this point, bothC3 convertases further assemble into C5 convertases (C4b2a3b andC3b3bBb). These complexes subsequently cleave complement component C5into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide(170 kDa). The C5a polypeptide binds to a 7 transmembrane G-proteincoupled receptor, which was originally associated with leukocytes and isnow known to be expressed on a variety of tissues including hepatocytesand neurons. The C5a molecule is the primary chemotactic component ofthe human complement system and can trigger a variety of biologicalresponses including leukocyte chemotaxis, smooth muscle contraction,activation of intracellular signal transduction pathways,neutrophil-endothelial adhesion, cytokine and lipid mediator release andoxidant formation.

The larger C5b fragment binds sequentially to later components of thecomplement cascade, C6, C7, C8 and C9 to form the C5b-9 membrane attackcomplex (“MAC”). The lipophilic C5b-9 MAC can directly lyseerythrocytes, and in greater quantities it is lytic for leukocytes anddamaging to tissues such as muscle, epithelial and endothelial cells. Insublytic amounts, the C5b-9 MAC can stimulate upregulation of adhesionmolecules, intracellular calcium increase and cytokine release. Inaddition, at sublytic concentrations the C5b-9 MAC can stimulate cellssuch as endothelial cells and platelets without causing cell lysis. Thenon-lytic effects of C5a and the C5b-9 MAC are comparable andinterchangeable.

Although the complement system has an important role in the maintenanceof health, it has the potential to cause or contribute to disease. Forexample, studies have shown that inhibition of the complement cascade ordepletion of complement components reduces damage in neurodegenerativediseases of the central nervous system or in experimental brain injury(see e.g., Feasby, T. E. et al. (1987) Brain Res. 419:97-103;Vriesendorp, F. J. et al. (1995) J. Neuroimmunol. 58:157-165; Jung, S.et al. (1995) Neurosci. Lett. 200:167-170; Dailey, A. T. et al. (1998)J. Neurosci. 18:6713-6722; Woodruff, T. M. et al. (2006) FASEB J.20:1407-1417; Leinhase, I. et al. (2006) BMS Neurosci. 14:7:55). Inparticular, rats deficient in C6, and thus unable to form the membraneattack complex (MAC), exhibit neither demyelination nor axonal damageand significantly reduced clinical score in the antibody-mediatedexperimental autoimmune encephalomyelitis (EAE) model for multiplesclerosis when compared with matched C6 sufficient rats (Mead, R. J. etal. (2002) J. Immunol. 168:458-465). However, levels of mononuclear cellinfiltration were equivalent to those seen in C6 sufficient rats. Meadet al. (2002) concluded that demyelination and axonal damage occur inthe presence of antibody and require activation of the entire complementcascade, including MAC deposition, which can be inhibited by depletionof C6.

Accordingly, reagents for inhibiting C6 are needed and are desirable fora variety of therapeutic purposes.

SUMMARY OF THE INVENTION

The invention provides anti-C6 antibodies having desirable functionalproperties for therapeutic purposes, including the ability toeffectively inhibit the functional activity of C6 such that formation ofthe membrane attack complex (MAC) is inhibited, both in vitro and invivo, as well as a very low K_(D) (e.g., 1×10⁻⁸ M, 1×10⁻⁹M, 5×10⁻¹⁰ M orless) and a very long half life (e.g., 40 hours or more). A panel of 38anti-human C6 antibodies was generated by immunizing rats with purifiedhuman C6 protein. Out of these 38, 2 were shown to inhibit MACformation. One particular rat anti-human C6 antibody having thesedesired functional properties was raised (referred to herein as 7E5),and a panel of humanized antibodies that retain the CDRs of 7E5 wasprepared, including the humanized mAbs 8G09, 7E12, 7G09, 8F07, 7F06,7F11, 7E11 and 7F02. The heavy and light chain variable regions of theseeight humanized mAbs, as well as the heavy chain variable region ofhumanized mAb 7C02 and the light chain variable region of humanized mAb7G08 were expressed in all 81 possible “mix and match” combinations, andall 81 pairings were demonstrated to effectively inhibit C6 functionalactivity. Furthermore, the epitope of 7E5 has been mapped within humanC6.

Accordingly, in one aspect, the invention pertains to an isolatedantibody that binds to human C6, wherein the antibody exhibits at leastthree of the following properties:

-   -   (a) has an IC₅₀ in a haemolytic assay of 0.5 μg/ml or less;    -   (b) has a K_(D), as determined by surface plasmon resonance, of        1×10⁻⁸ M or less;    -   (c) has an antibody-C6 binding half-life, as determined by        surface plasmon resonance, of 40 hours or greater; and    -   (d) cross-reacts with cynomolgus monkey C6.

Any combination of at least three of the above properties isencompassed. In one embodiment, the antibody exhibits properties (a),(b) and (c). In another embodiment, the antibody exhibits properties(a), (b), (c) and (d). In other embodiments, the antibody has a K_(D),as determined by surface plasmon resonance, of 1×10⁻⁹ M or less or5×10⁻¹⁰ M or less.

In another embodiment, the invention pertains to an isolated antibodythat binds to a region of human C6 containing all or a portion ofresidues 835-854 of SEQ ID NO: 52 (i.e., the antibody binds to one ormore residues within residues 835-854). In another embodiment, theantibody binds to an epitope that includes all or a portion of an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2 and SEQ ID NO: 3 (i.e., the antibody binds to one or more aminoacid residues within SEQ ID NO. 1, SEQ ID NO: 2 or SEQ ID NO: 3). Incertain embodiments, the epitope is part of a discontinuous epitoperecognized by the antibody. For example, in one embodiment, the antibodybinds an epitope that includes all or a portion of an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO: 3, wherein the epitope is discontinuous.

In yet another embodiment, the invention provides an antibody thatcross-competes for binding to human C6 with an antibody comprising aheavy chain variable region shown in SEQ ID NO: 5 and a light chainvariable region shown in SEQ ID NO: 10 (i.e., the heavy and light chainvariable regions of mAb 7E5). In yet other embodiments, the inventionprovides an antibody that cross-competes for binding to human C6 with amAb selected from the group consisting of 8G09, 7E12, 7G09, 8F07, 7F06,7F11, 7E11 and 7F02.

In various embodiments, an antibody of the invention is a human,humanized or chimeric antibody.

In various embodiments, an antibody of the invention comprises heavychain CDR1, 2 and 3 sequences shown in SEQ ID NOs: 6, 7 and 8,respectively, and comprises light chain CDR1, 2 and 3 sequences shown inSEQ ID NOs: 11, 12 and 13, respectively. For example, the antibody canbe a humanized antibody comprising the aforementioned CDRs.

In another aspect, the invention provides an isolated antibody thatbinds human C6, wherein the antibody comprises a heavy chain CDR3 shownin SEQ ID NO: 8; and a light chain CDR3 shown in SEQ ID NO: 13. Theantibody can further comprises a heavy chain CDR2 shown in SEQ ID NO: 7;and a light chain CDR2 shown in SEQ ID NO: 12. The antibody can furthercomprise a heavy chain CDR1 shown in SEQ ID NO: 6; and a light chainCDR1 shown in SEQ ID NO: 11. Representative examples of antibodiescomprising the aforementioned CDRs include the following:

-   -   (a) an antibody comprising the heavy chain variable region of        SEQ ID NO: 30 and the light chain variable region of SEQ ID NO:        31;    -   (b) an antibody comprising the heavy chain variable region of        SEQ ID NO: 32 and the light chain variable region of SEQ ID NO:        33;    -   (c) an antibody comprising the heavy chain variable region of        SEQ ID NO: 34 and the light chain variable region of SEQ ID NO:        35;    -   (d) an antibody comprising the heavy chain variable region of        SEQ ID NO: 36 and the light chain variable region of SEQ ID NO:        37;    -   (e) an antibody comprising the heavy chain variable region of        SEQ ID NO: 38 and the light chain variable region of SEQ ID NO:        39;    -   (f) an antibody comprising the heavy chain variable region of        SEQ ID NO: 40 and the light chain variable region of SEQ ID NO:        41;    -   (g) an antibody comprising the heavy chain variable region of        SEQ ID NO: 42 and the light chain variable region of SEQ ID NO:        43; and    -   (h) an antibody comprising the heavy chain variable region of        SEQ ID NO: 44 and the light chain variable region of SEQ ID NO:        45.

In yet another aspect, the invention provides an isolated antibody thatbinds human C6, which comprises:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence which is at least 90% identical to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 30,        32, 34, 36, 38, 40, 42, 44 and 46; and    -   (b) a light chain variable region comprising an amino acid        sequence which is at least 90% identical to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 31,        33, 35, 37, 39, 41, 43, 45 and 47.

In other embodiments, the heavy chain and light chain variable regionsare 95%, 96%, 97%, 98% or 99% identical to the aforementioned amino acidsequences. In another embodiment, the isolated antibody is one wherein:

(a) the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 30, 32, 34, 36, 38,40, 42, 44 and 46; and

(b) the light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, 39,41, 43, 45 and 47.

Expression vectors comprising a nucleotide sequence encoding thevariable region of a light chain, heavy chain, or both light and heavychains of an antibody of the invention, as well as host cellstransformed by such expression vectors, and methods for recombinantlyexpressing the antibody using the transformed host cells, are alsoencompassed by the invention. In one embodiment, an antibody of theinvention is expressed as a Fab fragment. In another embodiment, anantibody of the invention is expressed as a full-length antibody, suchas an IgG4 isotype antibody, such as with an IgG4 (S228P) constantregion.

Compositions comprising an antibody of the invention, such as apharmaceutical composition comprising an antibody of the invention and apharmaceutically acceptable carrier are also encompassed.

In another aspect, the invention pertains to methods of using anantibody of the invention. In one embodiment, the invention provides amethod of inhibiting MAC formation or activity in a subject, the methodcomprising administering to the subject the antibody of the invention inan amount effective to inhibit MAC formation or activity in the subject.In another embodiment, the invention provides a method of treating,preventing or reducing symptoms of a disorder mediated by undesiredactivity of the complement system in a subject, the method comprisingadministering to the subject an effective amount of an antibody of theinvention.

In another embodiment, the invention provides a method of regeneratingnerves in a subject, comprising administering to the subject atherapeutically effective amount of an antibody of the invention. In yetanother embodiment, the invention provides a method of promotingrecovery of damaged or degenerated nerves in a subject comprisingadministering to the subject a therapeutically effective amount of anantibody of the invention. In yet another embodiment, the inventionprovides a method of reducing or delaying degeneration of nerves in asubject comprising administering to the subject a therapeuticallyeffective amount of an antibody of the invention.

In one embodiment, the subject is suffering from a physical injury ofthe nerves, such as a traumatic injury (e.g., from an accident), asurgical injury or non-traumatic injury (e.g., a nerve compression). Inone embodiment, the injury is to the Peripheral Nervous System (PNS). Inanother embodiment, the injury is to the Central Nervous System (CNS).In one embodiment, the antibody is administered at or near the site ofinjury.

In another embodiment, the subject is suffering from an immune-mediatedinflammatory disorder or progressive neurodegenerative disorder. In oneembodiment, the disorder is acquired. In another embodiment, thedisorder is hereditary. In one embodiment, the disorder is a chronicdemyelinating neuropathy, such as multiple sclerosis (MS). In anotherembodiment, the disorder is a neurodegenerative disorder, such asmyasthenia gravis or amyotrophic lateral sclerosis (ALS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing the results of a haemolytic assay usingsupernatants from 38 hybridoma from two different rats immunized withhuman C6, demonstrating that supernatant 11 (producing the 7E5 mAb) hasthe strongest inhibitory effect.

FIG. 1B is a bar graph showing the results of a mannan-activatedcomplement ELISA using supernatants from 38 hybridoma from two differentrats immunized with human C6, demonstrating that supernatant 11(producing the 7E5 mAb) has the strongest inhibitory effect.

FIG. 2 is a graph showing the biacore kinetics for recombinant rat 7E5binding to human C6.

FIG. 3 shows the results for the epitope cross-blocking experimentbetween 27B1 mAb and 7E5 mAb, indicating that 7E5 occupies a differentepitope than 27B1.

FIG. 4A is an alignment of the human C6 partial amino acid sequence (SEQID NO: 50) and rat C6 partial amino acid sequence (SEQ ID NO: 51)showing the location of peptide 418.

FIG. 4B is a schematic diagram of the human C6 protein showing thelocation of peptide 418.

FIG. 5 is a bar graph showing that 7E5 blocks C6 in vivo in C6 deficientrats supplemented with human C6, as measured by haemolytic assay. Ratsreceived the indicated quantity of human C6 and antibody 7E5. Complementactivity is plotted on the Y-axis, whereby O.D. 1.0 indicates maximumlysis of sensitized erythrocytes, and O.D. 0 indicates absence of lysis.

FIG. 6A is an alignment of the amino acid sequences of the heavy chainvariable regions of rat anti-C5 7E5 mAb (SEQ ID NO: 5) and human VH3_1germline (SEQ ID NO: 48), with differences indicated. Indicated belowthe alignment are the amino acid exchanges targeted for humanization.

FIG. 6B is an alignment of the amino acid sequences of the light chainvariable regions of rat anti-C5 7E5 mAb (SEQ ID NO: 10) and human Vk2_5germline (SEQ ID NO: 49), with differences indicated. Indicated belowthe alignment are the amino acid exchanges targeted for humanization.

FIG. 7A is a bar graph showing the results of a haemolytic assaydemonstrating the inhibitory activity of all 81 possible combinations ofthe 9 humanized 7E5 variant VH chains and 9 humanized 7E5 variant VLchains (shown in FIGS. 7A and 7B, respectively).

FIG. 7B is a bar graph showing the results of a MAC ELISA assaydemonstrating the inhibitory activity of all 81 possible combinations ofthe 9 humanized 7E5 variant VH chains and 9 humanized 7E5 variant VLchains (shown in FIGS. 7A and 7B, respectively).

FIG. 8A is an alignment of the amino acid sequence of the 7E5 heavychain variable region (SEQ ID NO: 5) with the heavy chain amino acidsequences of the humanized 7E5 variants 7C02 (SEQ ID NO: 46), 7E11 (SEQID NO: 42), 7E12 (SEQ ID NO: 32), 7F02 (SEQ ID NO: 44), 7F06 (SEQ ID NO:38), 7F11 (SEQ ID NO: 40), 7G09 (SEQ ID NO: 34), 8F07 (SEQ ID NO: 36)and 8G09 (SEQ ID NO: 30). The conserved CDR1, 2 and 3 regions areindicated.

FIG. 8B is an alignment of the amino acid sequence of the 7E5 lightchain variable region (SEQ ID NO: 10) with the light chain amino acidsequences of the humanized 7E5 variants 7E11 (SEQ ID NO: 43), 7E12 (SEQID NO: 33), 7F02 (SEQ ID NO: 45), 7F06 (SEQ ID NO: 39), 7F11 (SEQ ID NO:41), 7G08 (SEQ ID NO: 47), 7G09 (SEQ ID NO: 35), 8F07 (SEQ ID NO: 37)and 8G09 (SEQ ID NO: 31). The conserved CDR1, 2 and 3 regions indicated.

FIG. 9 shows the affinity (Biacore) of 8 humanized F′Abs for human C6 incomparison to the affinity of the wild type 7E5 rat F′Ab.

FIG. 10 shows the results of an in Vivo nerve crush experiment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides anti-C6 antibodies that exhibitbeneficial functional properties. These functional features include, forexample: (a) an IC₅₀ in a haemolytic assay of 0.5 μg/ml or less; (b) aK_(D), as determined by surface plasmon resonance, of 5×10⁻¹⁰ M or less;(c) an antibody-C6 binding half-life, as determined by surface plasmonresonance, of 40 hours or greater; and/or (d) cross-reaction withcynomolgus monkey C6. In other embodiments, the antibodies includeparticular heavy and light chain variable regions and/or CDR sequences.For example, nine humanized heavy chain and nine humanized light chainvariable regions are provided that exhibit effective C6 inhibitoryactivity in all 81 possible “mix and match” combinations of the chains.In yet other embodiments, an anti-C6 antibody binds to the same epitopeas, or competes for bind to C6 with, a particular anti-C6 antibodydisclosed herein, such as 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11or 7F02.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “C6” (also referred to as “complement C6” or “complementcomponent C6”) refers to a component of the complement cascade thatbinds with the components C5b, C7, C8 and C9 to form the C5b-C9 membraneattack complex (MAC). The term “C6” includes any variants or isoforms ofC6 that are naturally expressed. Antibodies of the invention may bespecific for human CD6 and may not exhibit any cross-reactivity withother species. Alternatively, antibodies of the invention maycross-react with C6 from species other than human, such as cynomolgusmonkey. Alternatively, antibodies of the invention may cross-react withC6 from primates, such as cynomolgus monkey, but not cross-react withnon-primate C6, such as mouse or rat C6. C6, or any variants andisoforms thereof, may either be isolated from cells or tissues thatnaturally express them or be recombinantly produced using well-knowntechniques in the art. Genbank® (Accession No. NP_00110860.3) reportsthe amino acid sequence of human C6 as follows (SEQ ID NO:52):

  1 marrsvlyfi llnalinkgq acfcdhyawt qwtscsktcn sgtqsrhrqi vvdkyyqenf 61 ceqicskqet recnwqrcpi ncllgdfgpw sdcdpciekq skvrsvlrps qfggqpctap121 lvafqpcips klckieeadc knkfrcdsgr ciarklecng endcgdnsde rdcgrtkavc181 trkynpipsv qlmgngfhfl ageprgevld nsftggickt vkssrtsnpy rvpanlenvg241 fevqtaeddl ktdfykdlts lghnenqqgs fssqggssfs vpifysskrs eninhnsafk301 qaiqashkkd ssfirihkvm kvlnfttkak dlhlsdvflk alnhlpleyn salysrifdd361 fgthyftsgs lggvydllyq fsseelknsg lteeeakhcv rietkkrvlf akktkvehrc421 ttnklsekhe gsfiqgaeks islirggrse ygaalawekg ssgleektfs ewlesvkenp481 avidfelapi vdlvrnipca vtkrnnlrka lqeyaakfdp cqcapcpnng rptlsgtecl541 cvcqsgtyge ncekqspdyk snavdgqwgc wsswstcdat ykrsrtrecn npapqrggkr601 cegekrqeed ctfsimenng qpcinddeem kevdlpeiea dsgcpqpvpp engfirnekq661 lylvgedvei scltgfetvg ygyfrclpdg twrqgdvecq rtecikpvvq evltitpfqr721 lyrigesiel tcpkgfvvag psrytcqgns wtppisnslt cekdtltklk ghcqlgqkqs781 gsecicmspe edcshhsedl cvfdtdsndy ftspackfla ekclnnqqlh flhigscqdg841 rqlewglert rlssnstkke scgydtcydw ekcsastskc vcllppqcfk ggnqlycvkm901 gsstsektln icevgtirca nrkmeilhpg kcla

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., human C6). Such “fragments” are, for example between about 8 andabout 1500 amino acids in length, suitably between about 8 and about 745amino acids in length, suitably about 8 to about 300, for example about8 to about 200 amino acids, or about 10 to about 50 or 100 amino acidsin length. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and optional constant regions derived from humangermline immunoglobulin sequences. In one embodiment, human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant antibody,” as used herein, includes all chimeric,humanized and human antibodies that are prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for humanimmunoglobulin genes or a hybridoma prepared therefrom, (b) antibodiesisolated from a host cell transformed to express the antibody, e.g.,from a transfectoma, (c) antibodies isolated from a recombinant,combinatorial human or humanized antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.

The term “humanized antibody” refers to antibodies having frameworkregions from human germline sequences and CDRs from a non-human species(e.g., mouse, rat, rabbit) and includes, for example, antibodies inwhich the human framework regions and/or the CDRs have undergonespecific site directed mutagenesis to optimize binding. An exemplarydescription of the preparation of humanized anti-C6 antibodies isdescribed in Example 8.

The term “human antibody” includes antibodies having variable andconstant regions (if present) of human germline immunoglobulinsequences. Human antibodies of the invention can include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo) (see, Lonberg, N. et al. (1994) Nature368(6474): 856-859); Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). However, the term “human antibody” does notinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (i.e., humanized antibodies).

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to human C6 is substantially free of antibodies that specificallybind antigens other than human C6). An isolated antibody thatspecifically binds to an epitope of may, however, have cross-reactivityto other C6 proteins from different species. However, the antibodypreferably always binds to human C6. In addition, an isolated antibodyis typically substantially free of other cellular material and/orchemicals. In one embodiment of the invention, a combination of“isolated” antibodies having different C6 specificities is combined in awell-defined composition.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods for determiningwhat epitopes are bound by a given antibody (i.e., epitope mapping) arewell known in the art and include, for example, immunoblotting andimmunoprecipitation assays, wherein overlapping or contiguous peptidesfrom C6 are tested for reactivity with the given anti-C6 antibody.Methods of determining spatial conformation of epitopes includetechniques in the art and those described herein, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

The term “discontinuous epitope” refers to an epitope made up ofnoncontiguous amino acids. For example, the epitope may include residuesfrom multiple regions of human C6 which, when conformationally folded,are brought together (in proximity) such that the antibody binds to oneor more residues within each region.

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies compete for binding to an antigenand bind to the same, overlapping or encompassing continuous ordiscontinuous segments of amino acids. Those of skill in the artunderstand that the phrase “binds to the same epitope” does notnecessarily mean that the antibodies bind to exactly the same aminoacids. The precise amino acids to which the antibodies bind can differ.For example, a first antibody can bind to a segment of amino acids thatis completely encompassed by the segment of amino acids bound by asecond antibody. In another example, a first antibody binds one or moresegments of amino acids that significantly overlap the one or moresegments bound by the second antibody. For the purposes herein, suchantibodies are considered to “bind to the same epitope”.

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody bindswith an equilibrium dissociation constant (K_(D)) of approximately lessthan 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ Mor even lower when determined by surface plasmon resonance (SPR)technology in a BIACORE 2000 instrument using recombinant human C6 asthe analyte and the antibody as the ligand and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen”.

The term “K_(D)” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction. Typically, the antibodies of the invention bind to C6 witha dissociation equilibrium constant (K_(D)) of approximately 10⁻⁸ M orless, or 10⁻⁹ M or less, or 10⁻¹⁰ M or less or even lower whendetermined by surface plasmon resonance (SPR) technology in a BIACORE2000 instrument using recombinant human C6 as the analyte and theantibody as the ligand.

The term “kd” as used herein, is intended to refer to the off rateconstant for the dissociation of an antibody from the antibody/antigencomplex.

The term “ka” as used herein, is intended to refer to the on rateconstant for the association of an antibody with the antigen.

The term “IC₅₀” as used herein, refers to the concentration of anantibody or an antigen-binding portion thereof, which is needed, eitherin an in vitro or an in vivo assay, to inhibit a given biologicalresponse by half. That is, it is the half minimal (50%) inhibitoryconcentration (IC) of the antibody or antigen-binding portion thereof.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In oneembodiment, a human monoclonal antibody of the invention is of the IgG1isotype. In another embodiment, a human monoclonal antibody of theinvention is of the IgG2 isotype. In another embodiment, a humanmonoclonal antibody of the invention is of the IgG4 isotype. In anotherembodiment, a human monoclonal antibody of the invention is of the IgG4(S228P) isotype (i.e., an IgG4 isotype having a proline substitution ofthe wild type serine residue at amino acid position 228).

The term “binds to immobilized C6,” refers to the ability of a humanantibody of the invention to bind to C6, for example, expressed on thesurface of a cell or that is attached to a solid support.

The term “cross-reacts,” as used herein, refers to the ability of anantibody of the invention to bind to C6 from a different species. Forexample, an antibody of the present invention that binds human C6 mayalso bind another species of C6, such as cynomolgus monkey. As usedherein, cross-reactivity is measured by detecting a specific reactivitywith purified antigen in binding assays (e.g., SPR, ELISA) or bindingto, or otherwise functionally interacting with, cells physiologicallyexpressing C6. Methods for determining cross-reactivity include standardbinding assays as described herein, for example, by Biacore™ surfaceplasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument(Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that bind to C6, is intended to refer to a nucleic acidmolecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than C6, whichother sequences may naturally flank the nucleic acid in human genomicDNA. For example, SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26 and 28correspond to the nucleotide sequences encoding the heavy chain (V_(H))variable regions of anti-C6 monoclonal antibodies 8G09, 7E12, 7G09,8F07, 7F06, 7F11, 7E11 and 7F02, respectively. SEQ ID NOs: 15, 17, 19,21, 23, 25, 27 and 29 correspond to the nucleotide sequences encodingthe light chain (V_(H)) variable regions of anti-C6 monoclonalantibodies 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02,respectively.

The present invention also encompasses “conservative sequencemodifications” of the sequences set forth in SEQ ID NOs: 4-47, i.e.,nucleotide and amino acid sequence modifications which do not abrogatethe binding of the antibody encoded by the nucleotide sequence orcontaining the amino acid sequence, to the antigen. Such conservativesequence modifications include conservative nucleotide and amino acidsubstitutions, as well as, nucleotide and amino acid additions anddeletions. For example, modifications can be introduced into SEQ ID NOs:4-47 by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions include ones in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a human anti-C6 antibody is preferably replacedwith another amino acid residue from the same side chain family. Methodsof identifying nucleotide and amino acid conservative substitutionswhich do not eliminate antigen binding are well-known in the art (see,e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al.Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad.Sci. USA 94:412-417 (1997)).

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-C6 antibody coding sequence, suchas by saturation mutagenesis, and the resulting modified anti-C6antibodies can be screened for binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna. CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids or proteins of the invention may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form. A nucleic acid or protein is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of formation of the MAC by an anti-C6 antibody) areused interchangeably and encompass both partial and completeinhibition/blocking. The inhibition/blocking of C6 preferably reduces oralters the normal level or type of activity that occurs when C6 is notblocked or inhibited. Inhibition and blocking are also intended toinclude any measurable decrease in the binding or activity of C6 when incontact with an anti-C6 antibody as compared to C6 not in contact withan anti-C6 antibody, e.g., inhibits binding or activity of C6 by atleast about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In oneembodiment, the anti-C6 antibody inhibits binding or activity of C6 byat least about 70%. In another embodiment, the anti-C6 antibody inhibitsbinding or activity of C6 by at least 80%.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of suchtreatment, an antibody of the present invention, for example, a subjectin need such treatment.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an immune disorder. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Production of Antibodies to C6

The present invention encompasses antibodies, e.g., humanizedantibodies, that bind C6, e.g., human C6. Exemplary monoclonalantibodies that bind C6 include 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11,7E11 and 7F02, the heavy chain variable regions of which are shown inSEQ ID NOs: 5, 30, 32, 34, 36, 38, 40, 42 and 44, respectively, and thelight chain variable regions of which are shown in SEQ ID NOs: 10, 31,33, 35, 37, 39, 41, 43 and 45, respectively. The heavy chain CDR1, 2 and3 of these antibodies are shown in SEQ ID NOs: 6, 7 and 8, respectively,whereas the light chain CDR1, 2 and 3 of these antibodies are shown inSEQ ID NOs: 11, 12 and 13, respectively.

Monoclonal antibodies of the invention can be produced using a varietyof known techniques, such as the standard somatic cell hybridizationtechnique described by Kohler and Milstein, Nature 256: 495 (1975).Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies also canbe employed, e.g., viral or oncogenic transformation of B lymphocytes,phage display technique using libraries of human antibody genes andhumanization techniques such as those described in Example 6.

Accordingly, in one embodiment, a hybridoma method is used for producingan antibody that binds human C6. In this method, a rat, mouse or otherappropriate host animal can be immunized with a suitable antigen inorder to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the antigen used forimmunization. As described in Example 1, a particularly suitable hostanimal for raising anti-human C6 antibodies is a rat that is deficientin C6 (C6−/− rat), such that immunization with human C6 is will beconsidered completely “foreign.” Supernatants from immunized hostanimals can be tested in a suitable assay to detect anti-C6 activity,such as a haemolytic assay or MAC ELISA as described in detail inExample 1, to identify host animals expressing antibodies with C6inhibitory activity.

Lymphocytes from selected host animals can then be fused with myelomacells using a suitable fusing agent, such as polyethylene glycol, toform a hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). An exemplary fusionpartner is Y3-Ag1.2.3 cells, although other myeloma cells known in theart, such as SP2/0-Ag8.653 cells (ATCC, CRL 1580), are also suitable.Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen, suchas with the haemolytic assay and/or MAC ELISA. After hybridoma cells areidentified that produce antibodies of the desired specificity, affinity,and/or activity, the clones can be subcloned by limiting dilutionprocedures and grown by standard methods (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitableculture media for this purpose include, for example, D-MEM or RPMI-1640medium. In addition, the hybridoma cells may be grown in vivo as ascitestumors in an animal. The monoclonal antibodies secreted by the subclonescan be separated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. An exemplary,non-limiting example of preparation of hybridomas secreting anti-C6antibodies is described in detail in Example 1.

A non-human monoclonal antibody, such as a rat or mouse antibody, can behumanized using methods known in the art. For example, as described indetail in Example 6, a rat anti-human C6 mAb can be humanized using anapproach described in Hwang, W. Y et al. (2205) Methods 36:35-42. Thisapproach is based on the principle that if a non-human and a humanantibody have similarly structured CDRs, the human frameworks will alsosupport the non-human CDRs, with good retention of affinity. Thus, inthis method, the human framework sequences are chosen from the set ofhuman germline genes based on the structural similarity of the humanCDRs to those of the antibody to be humanized (same Chothia canonicalstructures). A phage display library of Fab variant sequences,containing deviating FR residues, is generated. After affinity-drivenselections, individual clones are screened for binding and off-rate andthe sequence human identity and homology is determined. Other approachesand methodologies for CDR-grafting and humanization that are wellestablished in the art also can be used to generate humanized anti-C6antibodies of the invention.

In another embodiment, antibodies and antibody portions that bind humanC6 can be isolated from antibody phage libraries generated using thetechniques described in, for example, McCafferty et al., Nature,348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991), Markset al., J. Mol. Biol., 222:581-597 (1991) and Hoet et al (2005) NatureBiotechnology 23, 344-348; U.S. Pat. Nos. 5,223,409; 5,403,484; and5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al. Additionally, production ofhigh affinity (nM range) human antibodies by chain shuffling (Marks etal., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)) may also be used.

In one embodiment, the antibody that binds human C6 is produced usingthe phage display technique described by Hoet et al., supra. Thistechnique involves the generation of a human Fab library having a uniquecombination of immunoglobulin sequences isolated from human donors andhaving synthetic diversity in the heavy-chain CDRs is generated. Thelibrary is then screened for Fabs that bind to human C6.

In one embodiment, antibodies directed against C6 are generated usingtransgenic or transchromosomal mice carrying parts of the human immunesystem rather than the mouse system. In one embodiment, the inventionemploys transgenic mice, referred to herein as “HuMAb mice” whichcontain a human immunoglobulin gene miniloci that encodes unrearrangedhuman heavy (μ and γ) and κ light chain immunoglobulin sequences,together with targeted mutations that inactivate the endogenous μ and κchain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856-859).Accordingly, the mice exhibit reduced expression of mouse IgM or κ, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994),supra; reviewed in Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci 764:536-546). The preparation of HuMAb mice is describedin Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen,J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al.(1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) NatureGenetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillonet al. (1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature368(6474): 856-859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology14: 845-851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharmInternational; U.S. Pat. No. 5,545,807 to Surani et al.; InternationalPublication Nos. WO 98/24884, published on Jun. 11, 1998; WO 94/25585,published Nov. 10, 1994; WO 93/1227, published Jun. 24, 1993; WO92/22645, published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchromosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto in the art as “KM mice”, are described in detail in PCT PublicationWO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-C6 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-C6 antibodies of the invention. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to in the art as “TC mice” can be used; suchmice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-C6antibodies of the invention.

Additional mouse systems described in the art for raising humanantibodies also can be applied to raising anti-C6 antibodies of theinvention, including but not limited to (i) the VelocImmune® mouse(Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavyand light chain variable regions have been replaced, via homologousrecombination, with human heavy and light chain variable regions,operatively linked to the endogenous mouse constant regions, such thatchimeric antibodies (human V/mouse C) are raised in the mice, and thensubsequently converted to fully human antibodies using standardrecombinant DNA techniques; and (ii) the MeMo® mouse (MerusBiopharmaceuticals, Inc.), in which the mouse contains unrearrangedhuman heavy chain variable regions but a single rearranged human commonlight chain variable region. Such mice, and use thereof to raiseantibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Generation of Transfectomas Producing Monoclonal Antibodies to C6

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202). Exemplary embodiments forrecombinant expression of anti-C6 antibodies are described further inExample 5 (in which pMQR expression vectors are used in HEK-293 hostcells), Example 6 (Fab expression using pCB4 expression vectors in E.coli host cells) and in Example 7 (CHO cell expression).

Furthermore, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by GS gene expression system disclosedin WO 87/04462, WO 89/01036 and EP 338 841 or other expression systemswell known in the art. The purified plasmid with the cloned antibodygenes can be introduced in eukaryotic host cells such as CHO-cells orNSO-cells or alternatively other eukaryotic cells like a plant derivedcells, fungi or yeast cells. The method used to introduce these genescould be methods described in the art such as electroporation,lipofectine, lipofectamine or other. After introducing these antibodygenes in the host cells, cells expressing the antibody can be identifiedand selected. These cells represent the transfectomas that can then beamplified for their expression level and upscaled to produce antibodies.Recombinant antibodies can be isolated and purified from these culturesupernatants and/or cells.

Alternatively these cloned antibody genes can be expressed in otherexpression systems such as E. coli or in complete organisms or can besynthetically expressed.

Cross-Competing and Same Epitope-Binding Antibodies

As described in detail in Example 3, the epitope to which the 7E5antibody binds has been mapped by alanine scanning mutagenesis toresidues within the region of human C6 corresponding to amino acids835-854 of SEQ ID NO: 52 (human C6). Particular peptides shown in SEQ IDNOs: 1, 2 and 3 also were shown to contain residues which form part ofthe epitope to which 7E5 binds. Accordingly, in one embodiment, theinvention provides an antibody that binds to an epitope of human C6 thatincludes all or a portion of residues 835-854 of SEQ ID NO: 52. Inanother embodiment, the invention provides an antibody that binds to anepitope of human C6 that includes all or a portion of residues 835-854of SEQ ID NO: 52, wherein the epitope is discontinuous. In anotherembodiment, the invention provides an antibody that binds to an epitopeof human C6 that includes all or a portion of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2 and 3. In anotherembodiment, the invention provides an antibody that binds to an epitopeof human C6 that includes all or a portion of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2 and 3, whereinthe epitope is discontinuous. In yet another embodiment, the inventionprovides an antibody that cross-competes for binding to human C6 with anantibody comprising a heavy chain variable region shown in SEQ ID NO: 5and a light chain variable region shown in SEQ ID NO: 10 (the VH and VLsequences of 7E5). In yet other embodiments, antibodies of the inventioncross-compete for binding to C6 with other anti-C6 antibodies describedherein, such as 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 or 7F02.

Such competing antibodies can be identified based on their ability tocompetitively inhibit binding to C6 of one or more of mAbs 7E5, 8G09,7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 in standard C6 binding assays.An exemplary assay for examining cross-competition for binding to C6 isan Epitope Sandwich ELISA, described in detail in Example 3.Furthermore, antibodies that recognize the same epitope or compete forbinding can be identified using other routine techniques. Suchtechniques include, for example, an immunoassay, which shows the abilityof one antibody to block the binding of another antibody to a targetantigen, i.e., a competitive binding assay. Competitive binding isdetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asC6. Numerous types of competitive binding assays are known, for example:solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay(see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA using1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990));and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77(1990)). Typically, such an assay involves the use of purified antigenbound to a solid surface or cells bearing either of these, an unlabeledtest immunoglobulin and a labeled reference immunoglobulin. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Usually, when acompeting antibody is present in excess, it will inhibit specificbinding of a reference antibody to a common antigen by at least 50-55%,55-60%, 60-65%, 65-70% 70-75% or more.

Accordingly, also, encompassed by the present invention are antibodiesthat bind to an epitope on C6 that comprises all or a portion of anepitope recognized by the particular antibodies described herein (e.g.,the same or an overlapping region or a region between or spanning theregion).

In one embodiment, the antibody that competes for binding to C6 and/orbinds to the same epitope on human C6 is a humanized antibody. Suchhumanized monoclonal antibodies can be prepared and isolated, forexample, as described in Example 6.

Other techniques for determining the epitope to which an antibody bindsinclude, for example, epitope mapping methods, such as, x-ray analysesof crystals of antigen:antibody complexes which provides atomicresolution of the epitope. Other methods monitor the binding of theantibody to antigen fragments or mutated variations of the antigen whereloss of binding due to a modification of an amino acid residue withinthe antigen sequence is often considered an indication of an epitopecomponent. In addition, computational combinatorial methods for epitopemapping can also be used. These methods rely on the ability of theantibody of interest to affinity isolate specific short peptides fromcombinatorial phage display peptide libraries. The peptides are thenregarded as leads for the definition of the epitope corresponding to theantibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

Once a single, archetypal anti-C6 mAb has been isolated that has thedesired properties described herein, it is straightforward to generateother mAbs with similar properties, e.g., having the same epitope, byusing art-known methods. For example, mice or rats may be immunized withC6 as described herein, hybridomas produced, and the resulting mAbsscreened for the ability to compete with the archetypal mAb for bindingto C6. Rats or mice can also be immunized with a smaller fragment of C6containing the epitope to which the archetypal mAb binds. The epitopecan be localized by, e.g., screening for binding to a series ofoverlapping peptides spanning C6. Alternatively, the method of Jesperset al., Biotechnology 12:899, 1994 may be used to guide the selection ofmAbs having the same epitope and therefore similar properties to thearchetypal mAb. Using phage display, first the heavy chain of thearchetypal antibody is paired with a repertoire of (preferably human)light chains to select a C6-binding mAb, and then the new light chain ispaired with a repertoire of (preferably human) heavy chains to select a(preferably human) C6-binding mAb having the same epitope as thearchetypal mAb. Alternatively variants of the archetypal mAb can beobtained by mutagenesis of cDNA encoding the heavy and light chains ofthe antibody.

Epitope mapping, e.g., as described in Champe et al. (1995) J. Biol.Chem. 270:1388-1394, can be performed to determine whether the antibodybinds an epitope of interest. “Alanine scanning mutagenesis,” asdescribed by Cunningham and Wells (1989) Science 244: 1081-1085, or someother form of point mutagenesis of amino acid residues in human C6 mayalso be used to determine the functional epitope for an anti-C6 antibodyof the present invention. Mutagenesis studies, however, may also revealamino acid residues that are crucial to the overall three-dimensionalstructure of C6 but that are not directly involved in antibody-antigencontacts, and thus other methods may be necessary to confirm afunctional epitope determined using this method.

The epitope bound by a specific antibody may also be determined byassessing binding of the antibody to peptides comprising fragments ofhuman C6. A series of overlapping peptides encompassing the sequence ofC6 may be synthesized and screened for binding, e.g. in a direct ELISA,a competitive ELISA (where the peptide is assessed for its ability toprevent binding of an antibody to C6 bound to a well of a microtiterplate), or on a chip. Such peptide screening methods may not be capableof detecting some discontinuous functional epitopes, i.e. functionalepitopes that involve amino acid residues that are not contiguous alongthe primary sequence of the C6 polypeptide chain.

The epitope bound by antibodies of the present invention may also bedetermined by structural methods, such as X-ray crystal structuredetermination (e.g., WO2005/044853), molecular modeling and nuclearmagnetic resonance (NMR) spectroscopy, including NMR determination ofthe H-D exchange rates of labile amide hydrogens in C6 when free andwhen bound in a complex with an antibody of interest (Zinn-Justin et al.(1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993)Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D 50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D 56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Use of Partial Antibody Sequences to Express Intact Antibodies

In certain embodiments, an anti-C6 antibody of the invention comprises aheavy chain CDR3 shown in SEQ ID NO: 8; and a light chain CDR3 shown inSEQ ID NO: 13. The antibody can further comprise a heavy chain CDR2shown in SEQ ID NO: 7; and a light chain CDR2 shown in SEQ ID NO: 12.The antibody can still further comprise a heavy chain CDR1 shown in SEQID NO: 6; and a light chain CDR1 shown in SEQ ID NO: 11. Exemplaryantibodies of the invention that utilize the aforementioned CDRs includethe following:

-   -   (a) an antibody comprising the heavy chain variable region of        SEQ ID NO: 30 and the light chain variable region of SEQ ID NO:        31;    -   (b) an antibody comprising the heavy chain variable region of        SEQ ID NO: 32 and the light chain variable region of SEQ ID NO:        33;    -   (c) an antibody comprising the heavy chain variable region of        SEQ ID NO: 34 and the light chain variable region of SEQ ID NO:        35;    -   (d) an antibody comprising the heavy chain variable region of        SEQ ID NO: 36 and the light chain variable region of SEQ ID NO:        37;    -   (e) an antibody comprising the heavy chain variable region of        SEQ ID NO: 38 and the light chain variable region of SEQ ID NO:        39;    -   (f) an antibody comprising the heavy chain variable region of        SEQ ID NO: 40 and the light chain variable region of SEQ ID NO:        41;    -   (g) an antibody comprising the heavy chain variable region of        SEQ ID NO: 42 and the light chain variable region of SEQ ID NO:        43; and    -   (h) an antibody comprising the heavy chain variable region of        SEQ ID NO: 44 and the light chain variable region of SEQ ID NO:        45.

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see PCT/US99/05535). Partial heavy and light chainsequence spanning the CDR regions is typically sufficient for thispurpose. The partial sequence is used to determine which germlinevariable and joining gene segments contributed to the recombinedantibody variable genes. The germline sequence is then used to fill inmissing portions of the variable regions. Heavy and light chain leadersequences are cleaved during protein maturation and do not contribute tothe properties of the final antibody. To add missing sequences, clonedcDNA sequences can be combined with synthetic oligonucleotides byligation or PCR amplification. Alternatively, the entire variable regioncan be synthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons.

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266:19867-19870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools, which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe AgeI site if the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains.

Plasmids for use in construction of expression vectors were constructedso that PCR amplified V heavy and V kappa light chain cDNA sequencescould be used to reconstruct complete heavy and light chain minigenes.These plasmids can be used to express completely human IgG₁κ or IgG₄κantibodies. Fully human and chimeric antibodies of the present inventionalso include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similarplasmids can be constructed for expression of other heavy chainisotypes, or for expression of antibodies comprising lambda lightchains.

Thus, in another aspect of the invention, structural features of anti-C6antibodies of the invention are used to create structurally relatedanti-C6 antibodies that retain at least one functional property of theantibodies of the invention, such as, for example,

-   -   (a) binds the same epitope as an anti-C6 antibody of the        invention, such as 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11        or 7F02;    -   (b) has an IC₅₀ in a haemolytic assay of 0.5 μg/ml or less;    -   (c) has a K_(D), as determined by surface plasmon resonance, of        1×10⁻⁸ M or less (or alternatively, 5×10⁻⁸ M or less, 1×10⁻⁹ M        or less, 5×10⁻⁹ M or less or 5×10⁻¹⁰ M or less);    -   (d) has an antibody-C6 binding half-life, as determined by        surface plasmon resonance, of 40 hours or greater; or    -   (e) cross-reacts with cynomolgus monkey C6.

In one embodiment, one or more CDR regions of antibodies of theinvention can be combined recombinantly with known framework regions andCDRs to create additional, recombinantly-engineered, anti-C6 antibodiesof the invention. The heavy and light chain variable framework regionscan be derived from the same or different antibody sequences. Theantibody sequences can be the sequences of naturally occurringantibodies or can be consensus sequences of several antibodies. SeeKettleborough et al., Protein Engineering 4:773 (1991); Kolbinger etal., Protein Engineering 6:971 (1993) and Carter et al., WO 92/22653.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-C6 antibody including: preparing an antibody including(1) heavy chain framework regions and heavy chain CDRs, where at leastone of the heavy chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 6, 7 and 8;and (2) light chain framework regions and light chain CDRs, where atleast one of the light chain CDRs includes an amino acid sequenceselected from the amino acid sequences of CDRs shown in SEQ ID NOs: 11,12 and 13; where the antibody retains the ability to bind to C6. Theability of the antibody to bind C6 can be determined using standardbinding and/or functional assays, such as those set forth in theExamples. Preferably, the antibody exhibits at least one, or at leasttwo or at least three or at least four or all five of the functionalproperties listed above as (a) through (e). Examples of such antibodies,as disclosed herein, include the 7E5, 8G09, 7E12, 7G09, 8F07, 7F06,7F11, 7E11 and 7F02 antibodies (as described in Example 6).

It is well known in the art that antibody heavy and light chain CDR3domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen (see, Hall et al., J.Imunol., 149:1605-1612 (1992); Polymenis et al., J. Immunol.,152:5318-5329 (1994); Jahn et al., Immunobiol., 193:400-419 (1995);Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., J.Mol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA,95:8910-8915 (1998); Barbas et al., J Am. Chem. Soc., 116:2161-2162(1994); Ditzel et al., J. Immunol., 157:739-749 (1996)). Accordingly,the recombinant antibodies of the invention prepared as set forth abovepreferably comprise the heavy and/or light chain CDR3 of the 7E5antibody, as set forth in SEQ ID NOs: 8 and 13, respectively. Examplesof such antibodies, as disclosed herein, include the 7E5, 8G09, 7E12,7G09, 8F07, 7F06, 7F11, 7E11 and 7F02 antibodies (as described inExample 6).

Moreover, in another embodiment, the invention further provides anti-C6antibodies comprising: (1) heavy chain framework regions, a heavy chainCDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region,wherein the heavy chain CDR3 region comprises the sequence of SEQ ID NO:8 and (2) light chain framework regions, a light chain CDR1 region, alight chain CDR2 region, and a light chain CDR3 region, wherein thelight chain CDR3 region comprises the sequence of SEQ ID NO: 13, whereinthe antibody binds C6. The antibody may further include the heavy chainCDR2 and/or the light chain CDR2 of the 7E5 antibody, as set forth inSEQ ID NOs: 7 and 12, respectively. The antibody may further comprisethe heavy chain CDR1 and/or the light chain CDR1 of the 7E5 antibody, asset forth in SEQ ID NOs: 6 and 11, respectively. Examples of suchantibodies, as disclosed herein, include the 7E5, 8G09, 7E12, 7G09,8F07, 7F06, 7F11, 7E11 and 7F02 antibodies (as described in Example 6).

Generation of Antibodies Having Modified Sequences

In another embodiment, the variable region sequences, or portionsthereof, of the anti-C6 antibodies of the invention are modified tocreate structurally related anti-C6 antibodies that retain binding(i.e., to the same epitope as the unmodified antibody) and, thus, arefunctionally equivalent. Methods for identifying residues that can bealtered without removing antigen binding are well-known in the art (see,e.g., Marks et al. (Biotechnology (1992) 10(7):779-83 (monoclonalantibodies diversification by shuffling light chain variable regions,then heavy chain variable regions with fixed CDR3 sequence changes),Jespers et al. (1994) Biotechnology 12(9):899-903 (selection of humanantibodies from phage display repertoires to a single epitope of anantigen), Sharon et al. (1986) PNAS USA 83(8):2628-31 (site-directedmutagenesis of an invariant amino acid residue at the variable-diversitysegments junction of an antibody); Casson et al. (1995) J. Immunol.155(12):5647-54 (evolution of loss and change of specificity resultingfrom random mutagenesis of an antibody heavy chain variable region).

Accordingly, in one aspect of the invention, the CDR1, 2, and/or 3regions of the engineered antibodies described above can comprise theexact amino acid sequence(s) as shown in SEQ ID NOs: 6-7 and 11-13 (7E5CDRs) as disclosed herein. However, in other aspects of the invention,the antibodies comprise derivatives from the exact CDR sequences of 7E5yet still retain the ability of to bind C6 effectively. Such sequencemodifications may include one or more amino acid additions, deletions,or substitutions, e.g., conservative sequence modifications as describedabove. Sequence modifications may also be based on the consensussequences described above for the particular CDR1, CDR2, and CDR3sequences of the 7E5 antibody.

Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of the 7E5 antibody (shown in SEQ IDNOs: 6-8 and 11-13). Ranges intermediate to the above-recited values,e.g., CDRs that are 90-95%, 95-98%, or 98-100% identical identity to oneor more of the above sequences are also intended to be encompassed bythe present invention.

In yet another embodiment, the invention provides an isolated antibodythat binds human C6, which comprises:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence which is at least 90% (or 90-95%, 95%-98%, 98%-100%,        95%, 96%, 97%, 98% or 99%) identical to an amino acid sequence        selected from the group consisting of SEQ ID NOs: 30, 32, 34,        36, 38, 40, 42, 44 and 46; and    -   (b) a light chain variable region comprising an amino acid        sequence which is at least 90% (or 90-95%, 95%-98%, 98%-100%,        95%, 96%, 97%, 98% or 99%) identical to an amino acid sequence        selected from the group consisting of SEQ ID NOs: 31, 33, 35,        37, 39, 41, 43, 45 and 47.

In yet another embodiment, the isolated antibody is one wherein

(a) the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 30, 32, 34, 36, 38,40, 42, 44 and 46; and

(b) the light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, 39,41, 43, 45 and 47.

Example 7 describes in detail “mix and match” experiments in which eachof these heavy and light chain variable regions were paired with eachother, in all 81 possible combinations, and the functional activity ofall 81 combinations in inhibiting C6 activity was demonstrated.

Furthermore, In another embodiment, one or more residues of a CDR may bealtered to modify binding to achieve a more favored on-rate of binding,a more favored off-rate of binding, or both, such that an idealizedbinding constant is achieved. Using this strategy, an antibody havingultra high binding affinity of, for example, 10¹⁰ M⁻¹ or more, can beachieved. Affinity maturation techniques, well known in the art andthose described herein, can be used to alter the CDR region(s) followedby screening of the resultant binding molecules for the desired changein binding. Accordingly, as CDR(s) are altered, changes in bindingaffinity as well as immunogenicity can be monitored and scored such thatan antibody optimized for the best combined binding and lowimmunogenicity are achieved.

Thus, for variable region modification within the VH and/or VL CDR1,CDR2 and/or CDR3 regions, site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedherein) are introduced. The mutations can be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the instant invention providesisolated anti-C6 monoclonal antibodies, or antigen binding portionsthereof, comprising: (a) a VH CDR1 region comprising an amino acidsequence shown in SEQ ID NO: 6 or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NO: 6; (b) a VH CDR2 region comprisingan amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NO: 7; (c) a VH CDR3 regioncomprising an amino acid sequence shown in SEQ ID NO: 8, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NO: 8; (d) aVL CDR1 region comprising an amino acid sequence shown in SEQ ID NO: 11,or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NO: 11;(e) a VL CDR2 region comprising an amino acid sequence shown in SEQ IDNO: 12, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNO: 11; and (f) a VL CDR3 region comprising an amino acid sequence shownin SEQ ID NO: 12, or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NO: 12.

In still another embodiment, the instant invention provides isolatedanti-C6 monoclonal antibodies, or antigen binding portions thereof,comprising: (a) a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 30, 32, 34,36, 38, 40, 42, 44 and 46, or an amino acid sequence having one, two,three, four, five, six, seven, eight, nine or ten amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 30, 32,34, 36, 38, 40, 42, 44 and 46; and (b) a light chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45 and 47, or an amino acidsequence having one, two, three, four, five, six, seven, eight, nine orten amino acid substitutions, deletions or additions as compared to SEQID NOs: 31, 33, 35, 37, 39, 41, 43, 45 and 47.

In addition to or instead of modifications within the CDRs,modifications can also be made within one or more of the frameworkregions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chainvariable regions of an antibody, so long as these modifications do noteliminate the binding affinity of the antibody. For example, one or morenon-germline amino acid residues in the framework regions of the heavyand/or the light chain variable region of a antibody of the invention,is substituted with a germline amino acid residue, i.e., thecorresponding amino acid residue in the human germline sequence for theheavy or the light chain variable region, which the antibody hassignificant sequence identity with. For example, an antibody chain canbe aligned to a germline antibody chain, which it shares significantsequence identity with, and the amino acid residues that do not matchbetween antibody framework sequence and the germline chain framework canbe substituted with corresponding residues from the germline sequence.When an amino acid differs between an antibody variable framework regionand an equivalent human germline sequence variable framework region, theantibody framework amino acid should usually be substituted by theequivalent human germline sequence amino acid if it is reasonablyexpected that the amino acid falls within one of the followingcategories:

(1) an amino acid residue which noncovalently binds antigen directly,

(2) an amino acid residue which is adjacent to a CDR region,

(3) an amino acid residue which otherwise interacts with a CDR region(e.g., is within about 3-6 Å of a CDR region as determined by computermodeling), or

(4) an amino acid reside which participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like. Accordingly, inone embodiment, an amino acid residue in the framework region of aantibody of the invention is substituted with the corresponding germlineamino acid residue which noncovalently binds antigen directly.

Residues which are “adjacent to a CDR region” include amino acidresidues in positions immediately adjacent to one or more of the CDRs inthe primary sequence of the antibody, for example, in positionsimmediately adjacent to a CDR as defined by Kabat, or a CDR as definedby Chothia (see e.g., Chothia and Lesk J. Mol. Biol. 196:901 (1987)).Accordingly, in one embodiment, an amino acid residue within theframework region of an antibody of the invention is substituted with acorresponding germline amino acid residue which is adjacent to a CDRregion.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. Such amino acids willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above. Accordingly, in one embodiment, an amino acid residuewithin the framework region of an antibody of the invention issubstituted with the corresponding germline amino acid residue whichotherwise interacts with a CDR region.

The amino acids at several positions in the framework are known to beimportant for determining CDR confirmation (e.g., capable of interactingwith the CDRs) in many antibodies (Chothia and Lesk, supra, Chothia etal., supra and Tramontano et al., J. Mol. Biol. 215:175 (1990), all ofwhich are incorporated herein by reference). These authors identifiedconserved framework residues important for CDR conformation by analysisof the structures of several known antibodies. The antibodies analyzedfell into a limited number of structural or “canonical” classes based onthe conformation of the CDRs. Conserved framework residues withinmembers of a canonical class are referred to as “canonical” residues.Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90,94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and94 of the heavy chain. Additional residues (e.g., CDRstructure-determining residues) can be identified according to themethodology of Martin and Thorton (1996)J. Mol. Biol. 263:800. Notably,the amino acids at positions 2, 48, 64 and 71 of the light chain and26-30, 71 and 94 of the heavy chain (numbering according to Kabat) areknown to be capable of interacting with the CDRs in many antibodies. Theamino acids at positions 35 in the light chain and 93 and 103 in theheavy chain are also likely to interact with the CDRs. Additionalresidues that may effect conformation of the CDRs can be identifiedaccording to the methodology of Foote and Winter (1992) J. Mol. Biol.224:487. Such residues are termed “vernier” residues and are thoseresidues in the framework region closely underlying (i.e., forming a“platform” under) the CDRs.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra.

Occasionally, there is some ambiguity about whether a particular aminoacid falls within one or more of the above-mentioned categories. In suchinstances, alternative variant antibodies are produced, one of which hasthat particular substitution, the other of which does not. Alternativevariant antibodies so produced can be tested in any of the assaysdescribed herein for the desired activity, and the preferred antibodyselected.

Additional candidates for substitution within the framework region areamino acids that are unusual or “rare” for an antibody at that position.These amino acids can be substituted with amino acids from theequivalent position of the human germline sequence or from theequivalent positions of more typical antibodies. For example,substitution may be desirable when the amino acid in a framework regionof the antibody is rare for that position and the corresponding aminoacid in the germline sequence is common for that position inimmunoglobulin sequences; or when the amino acid in the antibody is rarefor that position and the corresponding amino acid in the germlinesequence is also rare, relative to other sequences. It is contemplatedthat by replacing an unusual amino acid with an amino acid from thegermline sequence that happens to be typical for antibodies, theantibody may be made less immunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20%, preferably less than about 10%,more preferably less than about 5%, even more preferably less than about3%, even more preferably less than about 2% and even more preferablyless than about 1% of sequences in a representative sample of sequences,and the term “common”, as used herein, indicates an amino acid occurringin more than about 25% but usually more than about 50% of sequences in arepresentative sample. For example, all light and heavy chain variableregion sequences are respectively grouped into “subgroups” of sequencesthat are especially homologous to each other and have the same aminoacids at certain critical positions (Kabat et al., supra). When decidingwhether an amino acid in an antibody sequence is “rare” or “common”among sequences, it will often be preferable to consider only thosesequences in the same subgroup as the antibody sequence.

In general, the framework regions of antibodies are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human germline sequences from which they were derived. Ofcourse, many of the amino acids in the framework region make little orno direct contribution to the specificity or affinity of an antibody.Thus, many individual conservative substitutions of framework residuescan be tolerated without appreciable change of the specificity oraffinity of the resulting immunoglobulin. Thus, in one embodiment thevariable framework region of the antibody shares at least 85% sequenceidentity to a human germline variable framework region sequence orconsensus of such sequences. In another embodiment, the variableframework region of the antibody shares at least 90%, 95%, 96%, 97%, 98%or 99% sequence identity to a human germline variable framework regionsequence or consensus of such sequences.

Framework modifications can also be made to reduce immunogenicity of theantibody or to reduce or remove T cell epitopes that reside therein, asdescribed for instance by Carr et al in US2003/0153043.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation can contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues can be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043.

In addition to simply binding C6, an antibody may be selected for itsretention of other functional properties of antibodies of the invention,such as, for example: (a) binds the same epitope as an anti-C6 antibodyof the invention, such as 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11or 7F02;

-   -   (b) has an IC₅₀ in a haemolytic assay of 0.5 μg/ml or less;    -   (c) has a K_(D), as determined by surface plasmon resonance, of        5×10⁻¹⁰ M or less;    -   (d) has an antibody-C6 binding half-life, as determined by        surface plasmon resonance, of 40 hours or greater; or    -   (e) cross-reacts with cynomolgus monkey C6.        Additional Antibody Modifications

Antibodies of the present disclosure can contain one or moreglycosylation sites in either the light or heavy chain variable region.Such glycosylation sites may result in increased immunogenicity of theantibody or an alteration of the pK of the antibody due to alteredantigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Galaand Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-C6 antibodythat does not contain variable region glycosylation. This can beachieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

For example, in one embodiment, the glycosylation of an antibody ismodified, e.g., the variable region is altered to eliminate one or moreglycosylation sites resident in the variable region. More particularly,it is desirable in the sequence of the present antibodies to eliminatesites prone to glycosylation. This is achieved by altering theoccurrence of one or more N-X-(S/T) sequences that occur in the parentvariable region (where X is any amino acid residue), particularly bysubstituting the N residue and/or the S or T residue. In one embodiment,T95 is mutated to K95. In another embodiment, N47 is mutated to R47.

For example, aglycoslated antibodies can be made (i.e., which lackglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos.5,714,350 and 6,350,861.

Additionally or alternatively, the antibody can have an altered type ofglycosylation, such as a hypofucosylated antibody having reduced amountsof fucosyl residues or an antibody having increased bisecting GlcNacstructures. Such altered glycosylation patterns have been demonstratedto increase the ADCC ability of antibodies. Such carbohydratemodifications can be accomplished by, for example, expressing theantibody in a host cell with altered glycosylation machinery. Cells withaltered glycosylation machinery have been described in the art and canbe used as host cells in which to express recombinant antibodies of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (a (1,6)-fucosyltransferase), such thatantibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8^(−/−)cell lines were created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 and Yamane-Ohnuki et al. (2004) BiotechnolBioeng 87:614-22). As another example, EP 1,176,195 describes a cellline with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the α-1,6 bond-relatedenzyme. EP 1,176,195 also describes cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 describes a variant CHO cell line, Lec13 cells, withreduced ability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740).Antibodies with a modified glycosylation profile can also be produced inchicken eggs, as described in PCT Publication WO 06/089231.Alternatively, antibodies with a modified glycosylation profile can beproduced in plant cells, such as Lemna. Methods for production ofantibodies in a plant system are disclosed in the U.S. patentapplication corresponding to Alston & Bird LLP 60/836,998, filed on Aug.11, 2006. PCT Publication WO 99/54342 describes cell lines engineered toexpress glycoprotein-modifying glycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodiesexpressed in the engineered cell lines exhibit increased bisectingGlcNac structures which results in increased ADCC activity of theantibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody can be cleaved offusing a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removesfucosyl residues from antibodies (Tarentino et al. (1975) Biochem.14:5516-23).

Antibodies of the invention can be altered in the variable region toeliminate one or more glycosylation sites, and/or to improve physicalstability of the antibody. For example, in one embodiment, the physicalstability of the antibody is improved by substituting the serine atposition 228 of the variable region with a proline residue (i.e., theantibody has a variable region comprising a S228P mutation). The S228Palteration significantly stabilizes the antibody structure against theformation of intrachain disulfide bonds. In one embodiment, afull-length antibody of the invention is an IgG4 isotype with an S228Palteration (IgG4 S228P).

In another embodiment, the variable region is altered to eliminate oneor more glycosylation sites resident in the variable region. Moreparticularly, it is desirable in the sequence of the present antibodiesto eliminate sites prone to glycosylation. As described above, this canbe achieved by altering the occurrence of one or more N-X-(S/T)sequences that occur in the parent variable region (where X is any aminoacid residue), particularly by substituting the N residue and/or the Sor T residue. In one embodiment, T95 is mutated to K95. In anotherembodiment, N47 is mutated to R47.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention can be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. The antibody also can be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) ormodified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Each of these embodiments isdescribed in further detail below. The numbering of residues in the Fcregion is that of the EU index of Kabat.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcgR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcgRIII only, whereas monocytes express FcgRI, FcgRII andFcgRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch, J. V. and Kinet, J. P., Annu Rev. Immunol. 9(1991) 457-492. Non-limiting examples of in vitro assays to assess ADCCactivity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83(1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see Bruggemann, M. etal., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactiveassays methods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998)652-656. C1q binding assays may also be carried out to confirm that theantibody is unable to bind C1q and hence lacks CDC activity. See, e.g.,C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Toassess complement activation, a CDC assay may be performed (see, forexample, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996)163-171; Cragg, M. S. et al., Blood 101 (2003) 1045-1052; and Cragg, M.S, and M. J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and invivo clearance/half-life determinations can also be performed usingmethods known in the art (see, e.g., Petkova, S. B. et al., Int.Immunol. 18 (2006) 1759-1769).

In a particular embodiment, the antibody comprises a variable regionthat is mutated to improve the physical stability of the antibody. Inone embodiment, the antibody is an IgG4 isotype antibody comprising aSerine to Proline mutation at a position corresponding to position 228(S228P; EU index) in the hinge region of the heavy chain constantregion. This mutation has been reported to abolish the heterogeneity ofinter-heavy chain disulfide bridges in the hinge region (Angal et al.supra; position 241 is based on the Kabat numbering system). Forexample, in various embodiments, an anti-C6 antibody of the inventioncan comprise the heavy chain variable region of any of the antibodiesdescribed herein linked to a human IgG4 constant region in which theSerine at a position corresponding to position 241 as described in Angalet al., supra, has been mutated to Proline. Thus, for the heavy chainvariable regions linked to a human IgG4 constant region, this mutationcorresponds to an S228P mutation by the EU index.

In another embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, toincrease the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022. In still another approach,the antibody is modified to increase its biological half life byintroducing two mutations, one at a serine at position 434 and a secondmutation selected from a group consisting of: an isoleucine at position311, a valine at position 311, an isoleucine at position 436, and avaline at position 436. This approach is described in U.S. PatentPublication No. 2012/6128663.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another embodiment, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another embodiment, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutationsat positions 256, 290, 298, 333, 334 and 339 were shown to improvebinding to FcγRIII. Additionally, the following combination mutants wereshown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224Aand S298A/E333A/K334A.

In yet another embodiment, the Fc region is modified to reduce theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to reduce the affinity of the antibody for anFcγ receptor by introducing an amino acid substitution at positionPro329 and at least one additional amino acid substitution, preferablyselected from S228P, E233P, L234A, L235A, L235E, N297A, N297D, andP331S. This approach is described in U.S. Patent Publication No.2012/0251531.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

In addition, the antibody can be pegylated, for example, to increase thebiological (e.g., serum) half life of the antibody. To pegylate anantibody, the antibody, or fragment thereof, typically is reacted withpolyethylene glycol (PEG), such as a reactive ester or aldehydederivative of PEG, under conditions in which one or more PEG groupsbecome attached to the antibody or antibody fragment. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). As used herein, the term “polyethylene glycol”is intended to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401384.

Characterization of Monoclonal Antibodies to C6

Monoclonal antibodies of the invention can be characterized for bindingto C6 and/or functional inhibition of C6 using a variety of knowntechniques. Typically, binding of antibody to its target antigen isinitially characterized by ELISA. Briefly, microtiter plates can becoated with purified C6 in PBS, and then blocked with irrelevantproteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions ofplasma from C6-immunized mice are added to each well and incubated for1-2 hours at 37° C. The plates are washed with PBS/Tween 20 and thenincubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to alkaline phosphatase for 1 hour at 37° C. After washing,the plates are developed with ABTS substrate, and analyzed at OD of 405.Preferably, mice that develop the highest titers of antibodiesexhibiting the highest binding and/or functional inhibitory activity areused for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the C6 immunogen. Hybridomas that bind, preferably withhigh affinity, to C6 can then be subcloned and further characterized.One clone from each hybridoma, which retains the reactivity of theparent cells (by ELISA), can then be chosen for making a cell bank, andfor antibody purification.

Additionally or alternatively, a functional assay that determined theability of an antibody to inhibit or block C6 activity can be used forscreening and selection of an antibody of interest. Suitable in vitrofunctional assays include haemolytic assays and MAC ELISA assays, asdescribed in detail in Example 1. A suitable assay for determiningfunctional activity in vivo is described in detail in Example 4.

To purify anti-C6 antibodies, selected hybridomas can be grown in rollerbottles, two-liter spinner-flasks or other culture systems. Supernatantscan be filtered and concentrated before affinity chromatography withprotein A-Sepharose (Pharmacia, Piscataway, N.J.) to purify the protein.After buffer exchange to PBS, the concentration can be determined byOD₂₈₀ using 1.43 extinction coefficient or preferably by nephelometricanalysis. IgG can be checked by gel electrophoresis and by antigenspecific method.

To determine if the selected anti-C6 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated MAb bindingcan be detected with a streptavidin labeled probe. To determine theisotype of purified antibodies, isotype ELISAs can be performed usingart recognized techniques. For example, wells of microtiter plates canbe coated with 10 μg/ml of anti-Ig overnight at 4° C. After blockingwith 5% BSA, the plates are reacted with 10 μg/ml of monoclonalantibodies or purified isotype controls, at ambient temperature for twohours. The wells can then be reacted with either IgG1 or other isotypespecific conjugated probes. Plates are developed and analyzed asdescribed above.

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-C6 antibodies include standard assays known inthe art, for example, Biacore™ surface plasmon resonance (SPR) analysisusing a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden), asdescribed in Example 2 herein.

Preferably, an antibody of the invention binds to C6 with a K_(D) of5×10⁻⁸ M or less, binds to C6 with a K_(D) of 2×10⁻⁸ M or less, binds toC6 with a K_(D) of 5×10⁻⁹ M or less, binds to C6 with a K_(D) of 4×10⁻⁹M or less, binds to C6 with a K_(D) of 3×10⁻⁹ M or less, binds to C6with a K_(D) of 2×10⁻⁹ M or less, binds to C6 with a K_(D) of 1×10⁻⁹ Mor less, binds to C6 with a K_(D) of 5×10⁻¹⁰ M or less, or binds to C6with a K_(D) of 2.5×10⁻¹⁰ M or less.

Preferably, an antibody of the invention has a T_(1/2) (as determined bysurface plasmon resonance) of at least 24 hours, or at least 30 hours,or at least 36 hours, or at least 40 hours or at least 45 hours.

Antibody Physical Properties

Antibodies of this disclosure can be characterized by their variousphysical properties, to detect and/or differentiate different classesthereof.

In a preferred embodiment, the antibodies of the present disclosure donot contain asparagine isomerism sites. The deamidation of asparaginemay occur on N-G or D-G sequences and result in the creation of anisoaspartic acid residue that introduces a kink into the polypeptidechain and decreases its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-C6 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

In a preferred embodiment, antibodies are selected that do not degraderapidly. Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects, which can lead to the triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).Generally, it is preferred that the T_(M1) (the temperature of initialunfolding) be greater than 60° C., preferably greater than 65° C., evenmore preferably greater than 70° C. The melting point of an antibody canbe measured using differential scanning calorimetry (Chen et al (2003)Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) orcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

In one embodiment, an antibody of the invention has a high meltingtemperature. In one embodiment, the antibody has a melting point of atleast 65° C., more preferably, at least 66° C., even more preferably atleast 67° C., and even more preferably at least 68° C. Preferably anantibody of the invention has melting point in a range of 67° C. to 72°C., more preferably 68° C. to 72° C., or 69° C. to 72° C., or 70° C. to72° C. or 69° C. to 71.43° C.

II. Immunotoxins, Immunoconiugates and Antibody Derivatives

In another embodiment, the antibodies of the present invention arelinked to a therapeutic moiety, such as a cytotoxin, a drug or aradioisotope. When conjugated to a cytotoxin, these antibody conjugatesare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Conjugates of the antibody and a cytotoxin can be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate, iminothiolane,bifunctional derivatives of imidoesters such as dimethyl adipimidateHCL, active esters such as disuccinimidyl suberate, aldehydes such asglutaraldehyde, bis-azido compounds such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates such as toluene2,6-diisocyanate, and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). C¹⁴-labeled1-isothiocyanobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is a chelating agent suitable for conjugation of radionuclideto the antibody.

The toxin component of the immunotoxin can be, for example, achemotherapeutic agent, a toxin such as an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof, or asmall molecule toxin, or a radioactive isotope such as ²¹²Bi, ¹³¹I,¹³¹In, ¹¹¹In, ⁹⁰Y, and ¹⁸⁶Re.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates include the maytansinoids including DM-1 and DM-4,auristatins, adriamycin, doxorubicin, epirubicin, 5-fluorouracil,cytosine arabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan,cytoxin, taxoids, e.g. paclitaxel, and docetaxel, taxotere,methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide,ifosamide, mitomycin C, mitoxantrone, vincristine, vinorelbine,carboplatin, teniposide, daunomycin, carminomycin, aminopterin,dactinomycin, mitomycins, esperamicins, 5-FU, 6-thioguanine,6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, andother related nitrogen mustards. Also included are hormonal agents thatact to regulate or inhibit hormone action on tumors such as tamoxifenand onapristone. Toxins and fragments thereof which can be used includediphtheria A chain, nonbonding active fragments of diphtheria toxin,cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, PhytolacaAmericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, sapaonaria, officinalis inhibitor, gelonin,saporin, mitogellin, restrictocin, phenomycin, enomycin, and thetricothcenes. Small molecule toxins include, for example,calicheamicins, maytansinoids, palytoxin and CC1065.

Additional therapeutic agents which can be conjugated with the antibodyto form an immunotoxin include, antimetabolites, alkylating agents, DNAminor groove binders, DNA intercalators, DNA crosslinkers, histonedeacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In the conjugate, the antibody and therapeutic agent preferablyare conjugated via a linker cleavable such as a peptidyl, disulfide, orhydrazone linker. More preferably, the linker is a peptidyl linker suchas Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ IDNO: 15), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys,Cit, Ser, or Glu. The conjugates can be prepared as described in U.S.Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; andWO 08/103693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference.

Antibodies of the invention also can be used for diagnostic purposes,including sample testing and in vivo imaging, and for this purpose theantibody (or binding fragment thereof) can be conjugated to anappropriate detectable agent, to form an immunoconjugate. For diagnosticpurposes, appropriate agents are detectable labels that includeradioisotopes, for whole body imaging, and radioisotopes, enzymes,fluorescent labels and other suitable antibody tags for sample testing.

For C6 detection, the detectable labels can be any of the various typesused currently in the field of in vitro diagnostics, includingparticulate labels including metal sols such as colloidal gold, isotopessuch as I¹²⁵ or Tc⁹⁹ presented for instance with a peptidic chelatingagent of the N₂S₂, N₃S or N₄ type, chromophores including fluorescentmarkers, luminescent markers, phosphorescent markers and the like, aswell as enzyme labels that convert a given substrate to a detectablemarker, and polynucleotide tags that are revealed followingamplification such as by polymerase chain reaction. Suitable enzymelabels include horseradish peroxidase, alkaline phosphatase and thelike. For instance, the label can be the enzyme alkaline phosphatase,detected by measuring the presence or formation of chemiluminescencefollowing conversion of 1,2 dioxetane substrates such as adamantylmethoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-Star®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer is attached, they can bethe same or different molecules. In general, the number and/or type ofpolymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

Conjugation methods resulting in linkages which are substantially (ornearly) non-immunogenic are especially suited. Therefore, peptide- (i.e.amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, orether linkage are especially suited. These linkages are nearlynon-immunogenic and show reasonable stability within serum (see e.g.Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and the antibodydifferent conjugation strategies are at hand. In case the moiety isnaturally occurring or recombinant of between 50 to 500 amino acids,there are standard procedures in text books describing the chemistry forsynthesis of protein conjugates, which can be easily followed by theskilled artisan (see e.g. Hackenberger, C. P. R., and Schwarzer, D.,Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodimentthe reaction of a maleinimido moiety with a cysteine residue within theantibody or the moiety is used. This is an especially suited couplingchemistry in case e.g. a Fab or Fab′-fragment of an antibody is used.Alternatively in one embodiment coupling to the C-terminal end of theantibody or moiety is performed. C-terminal modification of a protein,e.g. of a Fab-fragment can e.g. be performed as described (Sunbul, M.and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; andProtease-catalyzed formation of C—N bonds is used by Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403).

Site specific reaction and covalent coupling can also be achieved by theselective reaction of terminal amino acids with appropriate modifyingreagents.

The reactivity of an N-terminal cysteine with benzonitrils (see Ren, H.et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used toachieve a site-specific covalent coupling.

Native chemical ligation can also rely on C-terminal cysteine residues(Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology(2009), 22 (Protein Engineering), 65-96).

EP 1 074 563 describes a conjugation method that is based on the fasterreaction of a cysteine within a stretch of negatively charged aminoacids with a cysteine located in a stretch of positively charged aminoacids.

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from un-bound components,like a labeled monovalent binder.

In one embodiment the effector moiety is selected from the groupconsisting of a binding moiety, a labeling moiety, and a biologicallyactive moiety

III. Compositions

In another embodiment, the present invention provides a composition,e.g., a composition, containing one or a combination of monoclonalantibodies of the present invention, formulated together with a carrier(e.g., a pharmaceutically acceptable carrier). Compositions containingbispecific molecules that comprise an antibody of the present inventionare also provided. In one embodiment, the compositions include acombination of multiple (e.g., two or more) isolated antibodies of theinvention. Preferably, each of the antibodies of the composition bindsto a distinct, pre-selected epitope of C6.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one or more additional therapeutic agents, such asanti-inflammatory agents, DMARDs (disease-modifying anti-rheumaticdrugs), immunosuppressive agents, and chemotherapeutics. Thepharmaceutical compositions of the invention can also be administered inconjunction with radiation therapy. Co-administration with otherantibodies is also encompassed by the invention.

As used herein, the terms “carrier” and “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Preferably,the carrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). Depending on the route of administration, the activecompound, i.e., antibody, bispecific and multispecific molecule, may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

Examples of adjuvants which may be used with the antibodies andconstructs of the present invention include: Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatised polysaccharides;polyphosphazenes; biodegradable microspheres; cytokines, such as GM-CSF,interleukin-2, -7, -12, and other like factors; 3D-MPL; CpGoligonucleotide; and monophosphoryl lipid A, for example 3-de-O-acylatedmonophosphoryl lipid A.

MPL adjuvants are available from Corixa Corporation (Seattle, Wash.;see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996.

Further alternative adjuvants include, for example, saponins, such asQuil A, or derivatives thereof, including QS21 and QS7 (AquilaBiopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; orGypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic,France); SAF (Chiron, Calif., United States); ISCOMS (CSL), MF-59(Chiron); the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium); Detox(Enhanzyn™) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs); polyoxyethyleneether adjuvants such as those described in WO 99/52549A1; syntheticimidazoquinolines such as imiquimod [S-26308, R-837], (Harrison, et al.,Vaccine 19: 1820-1826, 2001; and resiquimod [S-28463, R-848] (Vasilakos,et al., Cellular immunology 204: 64-74, 2000; Schiff bases of carbonylsand amines that are constitutively expressed on antigen presenting celland T-cell surfaces, such as tucaresol (Rhodes, J. et al., Nature 377:71-75, 1995); cytokine, chemokine and co-stimulatory molecules as eitherprotein or peptide, including for example pro-inflammatory cytokinessuch as Interferon, GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha andTGF-beta, Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15,IL-18 and IL-21, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13and other chemokine and co-stimulatory genes such as MCP-1, MIP-1 alpha,MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L; immunostimulatoryagents targeting ligands such as CTLA-4 and L-selectin, apoptosisstimulating proteins and peptides such as Fas; synthetic lipid basedadjuvants, such as vaxfectin, (Reyes et al., Vaccine 19: 3778-3786,2001) squalene, alpha-tocopherol, polysorbate 80, DOPC and cholesterol;endotoxin, [LPS], (Beutler, B., Current Opinion in Microbiology 3:23-30, 2000); ligands that trigger Toll receptors to produceTh1-inducing cytokines, such as synthetic Mycobacterial lipoproteins,Mycobacterial protein p19, peptidoglycan, teichoic acid and lipid A; andCT (cholera toxin, subunits A and B) and LT (heat labile enterotoxinfrom E. coli, subunits A and B), heat shock protein family (HSPs), andLLO (listeriolysin O; WO 01/72329). These and various further Toll-likeReceptor (TLR) agonists are described for example in Kanzler et al,Nature Medicine, May 2007, Vol 13, No 5.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent. Acceptablediluents include saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al. (1984) J. Neuroimmunol. 7:27).

Carriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the activecompound, use thereof in the pharmaceutical compositions of theinvention is contemplated. Supplementary active compounds can also beincorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the antibodies ofthe invention may be administered once or twice weekly by subcutaneousor intramuscular injection or once or twice monthly by subcutaneous orintramuscular injection.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for intravenous, intraperitoneal, oral, nasal,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the subject being treated, and the particular modeof administration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.001 percent to about ninety percent of active ingredient, preferablyfrom about 0.005 percent to about 70 percent, most preferably from about0.01 percent to about 30 percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.001 to 90% (morepreferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the invention will be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.It is preferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, preferably administered proximal tothe site of the target. If desired, the effective daily dose of atherapeutic composition may be administered as two, three, four, five,six or more sub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms. While it ispossible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the antibodies of the invention can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Thecomposition must be fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganismssuch as bacteria and fungi.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

IV. Uses and Methods of the Invention

The anti-C6 antibodies of the invention are able to functionally inhibitC6, both in vitro and in vivo, such that formation of MAC, whichrequires C6, is inhibited. Accordingly, in another aspect, the inventionpertains to a method of inhibiting MAC formation or activity in asubject, the method comprising administering to the subject the antibodyof the invention in an amount effective to inhibit MAC formation oractivity in the subject. In another embodiment, the invention provides amethod of treating, preventing or reducing symptoms of a disordermediated by undesired activity of the complement system in a subject,the method comprising administering to the subject an effective amountof an antibody of the invention. Examples of such disorders aredescribed further below.

As described in detail in U.S. Pat. No. 8,703,136 (the entire contentsof which are specifically incorporated herein by reference), it has beenestablished that axonal regeneration can be enhanced by inhibition ofthe complement system. Thus, use of the anti-C6 antibodies forinhibition of the complement system, and in particular inhibition of MACformation, can be used in the treatment of conditions that requireaxonal regeneration, e.g. in mammals affected by injury or disease ofthe central or peripheral nervous system. Conditions requiring axonalregeneration that may be treated in accordance with the inventioninclude physical injuries as well as neurodegenerative disorders of theperipheral or central nervous system.

In one embodiment, an antibody of the invention facilitates axonalregeneration. As used herein, the terms “facilitating axonalregeneration” or “facilitating nerve regeneration” are distinguishedfrom reducing or preventing axonal or nerve degeneration. Facilitation(or promotion) of axonal or nerve regeneration is herein understood tomean that regeneration of an axon or nerve is improved in subjects thatare treated as compared to non-treated subjects. Improved regenerationof an axon preferably is regeneration that occurs at an earlier point intime (after axonal or nerve injury or after start of the treatment) intreated subject as compared to non-treated subjects. Improvedregeneration of an axon or nerve may also comprise regeneration thatoccurs at a higher rate and/or to a larger extent in treated subject ascompared to non-treated subjects. An antibody according to the inventionthus preferably produces a gain of sensory or motor function.

Accordingly, in one embodiment, the invention provides a method ofregenerating nerves in a subject, comprising administering to thesubject a therapeutically effective amount of an antibody of theinvention. In another embodiment, the invention provides a method ofpromoting recovery of damaged or degenerated nerves in a subjectcomprising administering to the subject a therapeutically effectiveamount of the antibody of the invention. In yet another embodiment, theinvention provides a method of reducing or delaying degeneration ofnerves in a subject comprising administering to the subject atherapeutically effective amount of an antibody of the invention.

The subject can be suffering from a physical injury of the nerve, suchas an injury of the Peripheral Nervous System (PNS) or of the CentralNervous System (CNS), such as a nerve trauma from physical injury(discussed further below). The physical injury can be, for example, froma traumatic injury (e.g., an accident), a surgical injury or anon-traumatic injury (such as a nerve compression). In one embodiment,the antibody is administered at or near the site of injury.Alternatively, the subject may be suffering from a disease, such as animmune-mediated inflammatory disorder and/or progressiveneurodegenerative disorder, which may be acquired and/or hereditary,such as a chronic demyelinating neuropathy, such as multiple sclerosis(MS), or other neurodegenerative disorder such as myasthenia gravis oramyotrophic lateral sclerosis (ALS) (discussed further below).

Improvement in axonal regeneration is preferably determined byfunctional tests that are relatively easily conducted in human subjects,e.g. recovery of sensory or motor function is preferably determined in astandardized test as is available in the art (see e.g. Wong, K. H et al.(2006) Scand. J. Plast. Reconstr. Surg. Hand Surg. 40:219-224;Jerosch-Herold (2005) Hand Surg. 30:252-264. Suitable tests preferablyare quantitative, standardized and more preferably have had theirpsychometric properties evaluated and quantified. Such tests includee.g. the Weinstein Enhanced Sensory Test (WEST) or the Semmes-WeinsteinMonofilament Test (SWMT) and the shape-texture identification (STI) testfor tactile gnosis. Improved axonal regeneration may experimentally bedetermined in test animals by functional tests for recovery of sensoryor motor function as described by Hare, G. M. T et al. (1992) Plasticand Reconstr. Surg. 89:251-258 and De Koning, P. et al. (1986) J.Neurol. Sci. 74:237-246. An antibody preferably produces a gain ofsensory or motor function, as may be determined in e.g. anabove-indicated test.

Example 8 describes in detail an animal model that can be used to testthe effect of anti-C6 antibodies of the invention on sensory function.This nerve crush model (crush of the nervus ischiadicus) is used to testthe effect of anti-human C6 monoclonal antibodies on the recovery ofsensory function in C6-knock-out rats (PVC) supplemented with human C6.The nerve crush is a model for peripheral nerve injury. See WO2010/005310 (PCT/NL2009/050418); and de Jonge et al (2004) Hum MolGenet. 13(3):295-302.

Improved axonal regeneration may also be experimentally determined intest animals by histological examination, e.g. improved remyelinationmay be determined by comparing measurements of myelin sheaths around theaxon in treated animals vs. non-treated animals, whereby a thickermyelin sheath is indicative of improved remyelination. More efficientaxonal regeneration may be determined as the production of single, largediameter, axon sprouts in treated animals as compared to clusters ofsmaller axons in non-treated animals.

The appropriate dose of an antibody is that amount effective to promoteaxonal regeneration as may be seen by improvement of sensory or motorfunction as described above. By “effective amount,” “therapeuticamount,” or “effective dose” is meant that amount sufficient to elicitthe desired pharmacological or therapeutic effects, thus resulting ineffective treatment of the injury or disorder.

In order to minimize nerve injury and/or to facilitate axonalregeneration at soon as possible, in the methods of the invention, theantibody is preferably administered shortly after the occurrence of thenerve injury, i.e. within 24, 12, 6, 3, 2, or 1 hours, more preferablywithin 45, 30, 20 or 10 minutes after the occurrence of the nerveinjury. In one embodiment of the invention, the antibody may beadministered (e.g. as a precautionary measure) prior to surgery with arisk of nerve injury (see below), so as to minimize nerve injury and/orto facilitate axonal regeneration immediately upon surgical injury ofthe nerve.

A variety of conditions that require axonal regeneration may be treatedwith the antibodies of the invention. The conditions include injury ofthe PNS, as well as injury of the CNS. The conditions include nervetrauma as a result of physical injuries as well as resulting fromdisease. Such diseases include immune-mediated inflammatory disorders orinjuries and/or progressive neurodegenerative disorders which may beacquired and/or hereditary.

The physical injuries of the PNS and CNS may be traumatic injuries,including surgical injuries, or non-traumatic injuries. Traumatic PNSand CNS injuries that may be treated with the methods and/or themedicaments of the invention include spinal cord lesions, as well astraumatic wounds to peripheral nerves, including injuries fromcollisions, motor vehicle accidents, gun wounds, fractures,dislocations, lacerations, or some other form of penetrating trauma.Peripheral nerves injured through trauma that may be treated include thedigital, median, ulnar, radial, facial, spinal accessory and brachialplexus nerves.

Surgical PNS injuries are herein understood as injuries to peripheralnerves that arise when it becomes clinically necessary to remove ordissect a nerve during a surgical procedure. This occurs in thousands ofsurgical procedures each year. One example of surgically injuredperipheral nerves that may be treated with the methods and/ormedicaments of the invention include e.g. the cavernous nerves thatsupport erectile function and bladder control; these nerves are oftendamaged during surgical removal of a prostate tumour and the tissuearound it. Another example of a surgically injured peripheral nerve thatmay be treated in accordance with the invention is the phrenic nerveafter coronary artery bypass grafting (CABG).

Non-traumatic physical PNS injuries that may be treated with theantibodies of the invention include compression and/or adhesion ofperipheral nerves, also known as entrapment syndromes. The most commonentrapment syndrome is carpal tunnel syndrome.

In addition, immune-mediated inflammatory disorders or injuries may betreated with the antibodies of the invention. These includedemyelinating diseases of the central and peripheral nervous systemsthat are believed to have an autoimmune basis and result in nervedemyelination as a result of damage caused to oligodendrocytes or tomyelin directly. Such demyelinating diseases include e.g. Guillain-Barresyndrome (GBS; also referred to as inflammatory demyelinatingpolyneuropathy, acute idiopathic polyradiculoneuritis, acute idiopathicpolyneuritis, French Polio and Landry's ascending paralysis).Preferably, of the invention antibodies are applied to promote axonalregeneration subsequent to acute phase in GBS. Similarly chronicinflammatory demyelinating polyneuropathy (CIDP), considered the chroniccounterpart of GBS, may be treated with the antibodies of the invention.Multiple sclerosis (MS) is another demyelinating disease that may betreated with the antibodies of the invention.

Further neurodegenerative CNS and/or PNS disorders with a geneticcomponent that may be treated with the antibodies of the inventioninclude Amyotrophic Lateral Sclerosis (ALS, sometimes called LouGehrig's disease), Charcot-Marie-Tooth disease (Hereditary Motor andSensory Neuropathy, HMSN) and Huntington Disease (HD).

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1: Generation of Rat Anti-Human C6 MonoclonalAntibodies

Rat anti-human C6 monoclonal antibodies were generated by immunizingfive rats of the PVG C6−/− strain with human C6 protein. C6 deficientrats were chosen because according to the current understanding in thefield it is extremely difficult to generate functional C6 antibodies innormal rodents. It is hypothesized that immunization against C6 is notefficient in wild type animals due to the high degree of homology of C6protein between humans and rodents. The antibody response in C6deficient animals is more robust because these animals have nofunctional C6 protein in circulation and thus are likely to consider C6as completely “foreign.” Human C6 was purified from whole human serum bymeans of affinity chromatography using 23D1 mouse monoclonal antibody23D1 (described in detail in L. Clayton (2005) Ph.D. Thesis, CardiffUniversity) coupled to Sepharose (GE Healthcare Cat No. 17-0717-01).

Antigen and Immunization:

One week before immunization, a pre-immunization bleed was performed onthe rats by collecting 100 μl of blood from the tail vein. On Day 1 ofthe immunization, rats were injected at four locations subcutaneously(s.c.) with 100 μg C6 antigen in Complete Freund's Adjuvant (CFA), in avolume of 250 μl per injection. Booster injections were performed onDays 14 and 21, again at four s.c. locations with 50 μg C6 antigen inIncomplete Freund's Adjuvant (IFA) in a volume of 250 μl per injection.Test bleeds were performed on Day 36 by collecting 100 μl of blood fromthe tail vein for in vitro tests. These test bleeds were analyzed in aC6 ELISA, a C6 Western blot and in a haemolytic assay (described furtherbelow), which showed that all five rats had a positive immune responseagainst human C6: all five rats had antibodies that blocked hemolysis inthe haemolytic assay and all five rats had antibodies that recognizedpurified C6 on Western blot (denaturing conditions). A pre-fusionbooster was performed on Day 62 by injection of 100 μg antigen in 250 μlPBS intraperitoneally. Finally, a pre-fusion booster was performed onDay 64 by injection of 100 μg antigen in 250 μl PBS intravenously (tailvein). Spleens from two rats were harvested on Day 66 (with the otherthree rats left as backup) and the isolated splenocytes used forhybridoma preparation.

Hybridoma Preparation:

Hybridomas were prepared by fusion of the splenocytes from the humanC6-immunized rats with Y3-Ag1.2.3 fusion partner cells using standardpolyethylene glycol (PEG)-mediated fusion essentially as described inLuk, J. M. et al. (1990) J. Immunol. Methods 129:243-250. Supernatantswere harvested and used for initial screening for anti-human C6antibodies via ELISA using 96-well plates coated with human C6 antigen.Positive clones were selected and subcloned. Thirty-eight positiveclones were selected for further analysis.

Haemolytic Assay:

These 38 supernatants and control supernatants were further tested in ahaemolytic assay at a 1:50 dilution using human serum as complementsource. In this assay, erythrocytes coated with complement activatingantigens are incubated in the presence of serum. The serum contains thecomponents of the complement system, which are activated through theclassical pathway when the coated erythrocytes are encountered. MAC isformed as part of the terminal complement system and MAC initiates lysisof the erythrocytes. Erythrocyte lysis can be quantified by measuringthe OD at 405 or 415 nm in the supernatant and is a direct measurementof the activity of the MAC. Complement inhibitors can be tested in thissystem because if they are effective they will prevent erythrocyte lysisin a quantitative fashion.

To perform the assay, a haemolytic system ready to use was obtainedcommercially (Virion/Serion GmbH, Wurzburg, Gemany) along with CFTbuffer (Virion/Serion GmbH, Wurzburg, Gemany). The CFT buffer wasprepared according to the manufacturer's instructions. The haemolyticsystem was placed on a rollerbank in a coldroom to thoroughly mix theerythrocytes. To prepare a CFT serum cocktail, 100 μl of human serum wasadded to 5 ml of CFT buffer. Dilutions of test inhibitors, in a volumeof 50 μl, were added to round bottom 96-well plates, 50 μl of CFT serumcocktail was added to each well and mixed carefully while pipetting andthe plates were incubated at 37° C. for 30 minutes. Positive controlswas EDTA. Negative control was serum free or C6 deficient serum. Afterincubation, plates were spun down at 2000 rpm for 5 minutes (Hettichtable top centrifuge) and 80 μl of supernatant was transferred to flatbottom plate for measurement at 405 or 415 nm. The OD was measuredwithin 10 minutes of transfer.

Test supernatants were added in dilution in the haemolytic assay todetermine whether they prevent erythrocyte lysis. Exemplary results areshown in FIG. 1A, which demonstrates that certain of the supernatantsexhibited stronger inhibitory activity than others. In particular,supernatants #6-12 exhibited stronger inhibition than the othersupernatants, with supernatants #11 and #12 showing the strongestinhibition. The supernatants (1:50 dilution) were also tested in thehaemolytic assay using rat serum as complement source and no inhibitoryeffect was observed, demonstrating that the inhibitory activity of theantibodies was specific for human C6.

MAC ELISA Assay:

A second assay was used to determine whether the supernatants were ableto block MAC formation. In this assay, the ELISA wells in the plate arecoated with either Mannan or IgG as trigger for either the Lectin or theClassical pathway of complement, respectively, in the presence of serum.The serum contains the components of the complement system, which areactivated through either pathway when they are exposed to the coatedplate. The MAC is formed as part of the terminal complement system andMAC will be deposited on the ELISA plate. MAC deposition on the platecan be detected by HRP-conjugated antibodies and visualized by enzymaticreaction in the presence of a chromogen and substrate. This reactionproduces a color that can be quantified by measuring the OD at 450 or655 nm. The OD is a direct measurement of the amount of MAC formation.Complement inhibitors can be tested in this system because if they areeffective they will prevent or inhibit deposition of MAC on the plate.

In the second assay used to test the hybridoma supernatants, a mannanactivated complement ELISA assay was done. Briefly, ELISA plates werecoated with mannan and diluted hybridoma supernatant and human serum wasadded. Complement components that form a complex on the mannan coatedplate can be detected using antibodies. In this particular assay, C9 wasdetected as an indicator of MAC formation. If less C9 is detected in thepresence of the supernatant versus the absence of the supernatant, thisindicates MAC inhibition. Positive controls used were EDTA (since thereaction is calcium dependent).

To perform the assay, coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, 15 mMNaN₃, pH 9.6), blocking buffer (1 mg/ml BSA/HAS, 10 mM Tris/HCl, pH 7.4,145 mM NaCl, 15 nM NaN₃, pH 7.4) wash buffer (1×TBS, 0.05% Tween 20, 5mM CaCl₂) and dilution buffer (4 mM barbital, 145 mM NaCl, 2 mM CaCl₂, 1mM MgCl₂, 0.3% BSA, 0.02% Tween 20) were prepared. The wells of aflat-bottomed high binding 96-well plate were coated with 100 μl ofcoating buffer containing 10 μg/ml mannan (Sigma, Cat. No. M7504) andincubated overnight at 4° C. Plates were blocked with 200 μl blockingbuffer for 1 hour at room temperature. Human serum in dilution buffer(1:100) was diluted with supernatant (1:50) in round bottom plates and50 μl per well was added to the flat bottom high binding plates. Theplates were incubated for 1 hour at 37° C., followed by washing 3 timeswith wash buffer. Anti-C5b-9neo (clone aE11; DAKO, Cat. No. M0777) wasdiluted 1:100 in dilution buffer, 50 μl was added per well and theplates were incubated for 1 hour at room temperature, followed bywashing 3 times with wash buffer. Anti-mouse HRP (DAKO, Cat. No. P0447)was diluted 1:2000 in dilution buffer, 50 μl was added per well and theplates were incubated for 30 minutes at room temperature, followed bywashing three times with wash buffer. To develop, 50 μl TMB chromogen(TMB: Sigma T2885; stock solution prepared of 10 mg/ml TMB in DMSO) and10 μl 3% H2O2 was added to 5 ml NaAc buffer (8.2 gm Natrium Acetate, 21gm Citric Acid Monohydrate in 1 liter H₂O) and distributed to the96-well plates. The reaction was stopped with 25 μl 1 M H₂SO₄ and the ODwas measured with a spectrophotometer at 450 nm/655 nm.

Exemplary results of this assay are shown in FIG. 1B, which demonstratesthat two supernatants, #11 and #17 had significantly better inhibitoryability than the other 36 supernatants, with supernatant #11 having byfar the most superior inhibitory ability of all of the clones analyzed.

Since supernatant #11 exhibited the strongest inhibition in both thehaemolytic assay and in the MAC ELISA assay, this hybridoma was selectedfor further characterization. The monoclonal antibody produced by thishybridoma is referred to herein as 7E5.

Example 2: Characterization of 7E5 Monoclonal Antibody

In this example, additional experiments were performed to furtherexamine the binding and functional characteristics of the rat anti-humanC6 monoclonal antibody 7E5.

Cross-Reactivity:

Western blots were performed using human serum and serum from cynomolgusmonkeys (Cyno). Human and Cyno serum was used for PAGE (10% gel) andstandard Western blotting. Antibody was incubated on the blots for 1hour in a 1:500 dilution. Detection was done using anti-rat horse radishperoxidase (HRP) (DAKO, 1:1000) and Lumilight (Roche) in a LAS3000(Fuji) darkbox imaging system. The results indicated that 7E5 is able torecognize both human and cynomolgus monkey C6.

Binding Kinetics:

To investigate kinetics of 7E5 binding to C6, surface plasmon resonancemeasurement in a BIACORE 2000 (GE Healthcare) equipped with aresearch-grade CM5 sensor chip was used. The ligand (C6, 113 kDa) wasimmobilized using amine-coupling chemistry. The surface of flow cell twowas activated for 7 minutes with a 1:1 mixture of 0.1 M NHS(N-hydroxysuccinimide) and 0.4 M EDC (3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide) at a flow rate of 5 μl/min. The ligand at aconcentration of 10 μg/ml in 10 mM sodium acetate, pH 5.0, wasimmobilized at a density of 955 RU. The surface was blocked with a 7minute injection of 1 M ethanolamine, pH 8.0.

Flow cell 1 was immobilized with an antibody from an earlier experiment(αvWWF; 987 RU) and served as a reference surface.

To collect kinetic binding data, the analytes (anti C6-antibodies, 150kDa) in 10 mM HEPES, 150 mM NaCl, 0.005% P20, pH 7.4, were injected overthe two flow cells at a flow rate of 30 μl/min and at a temperature of25° C. The injected concentrations differ per antibody. Data werecollected at a rate of 1 Hz. The complex was allowed to associate anddissociate for 90 and 300 seconds, respectively. The surfaces wereregenerated with a 10 second injection of 0.1 M HCl. Duplicateinjections (in random order) of each sample and a buffer blank wereflowed over the two surfaces.

The data were fit to a simple 1:1 interaction model using the globaldata analysis option available within BiaEvaluation 4.1 software. TheBiacore kinetic results are shown in FIG. 2. Results from arepresentative experiment also are summarized in Tables 1-4 below.

TABLE 1 Kinetics of 7E5 Binding Determined by Surface Plasmon Resonanceka 1.69 × 10⁴M−1s−1 kd 4.27 × 10⁻⁶ s−1    K_(D) 2.53 × 10⁻¹⁰M     Rmax36 RU Rmax 95%

TABLE 2 Complex Half Life for 7E5 Binding kd (s−1) t_(1/2) (sec) t_(1/2)(min) t_(1/2) (hour) 4.27 × 10⁻⁶ s−1 162330 2705.5 45.1

TABLE 3 Time to 5% Dissociation for 7E5 Binding kd (s−1) Time (min) Time(hour) R (RU) 4.27 x 10⁻⁶ s−1 200.2 3.3 34

TABLE 4 Time to 95% Dissociation for 7E5 Binding kd (s−1) Time (min)Time (hour) R (RU) 4.27 × 10⁻⁶ s−1 11692.9 194.9 2

The K_(D) of 7E5 is calculated as 2.5×10⁻¹⁰ M. This high affinity isprimarily caused by the high antibody-antigen complex half-life (45hrs). Thus, 7E5 binding is very stable, with the half-life of the 7E5-C6complex estimated at over 40 hrs.

To determine whether the antigen-antibody complex could be released inthe endosomes and lysosomes after binding and cellular uptake by the Fcgamma receptor, the sensitivity of the 7E5-C6 complex was tested in lowpH. Since in lysosomes the pH is about 4.8, complex stability was testedup to pH=4. In this BIACORE experiment the 7E5-C6 complex on the chipwas washed with buffers with decreasing pH. Hepes buffered saline ((HBS)was used for pH 7.4, 7.0 and 6.5. 10 mM Sodium Acetate was used for pH6.0, 5.5, 5.0, 4.5 and 4.0. It was observed that the stability of thecomplex is not sensitive for low pH.

Effect of Pre-Incubation on Haemolytic Assay:

Since BIACORE experiments revealed that the slow release of 7E5 from C6is a primary determinant of the K_(D) of 7E5, it was investigatedwhether pre-incubation of 7E5 with the complement source (human serum)prior to adding the erythrocytes increased inhibitory efficacy in thehaemolytic assay. 7E5 was pre-incubated with human serum for 30, 90 or180 minutes at room temperature (20° C.) before the erythrocytes wereadded and the reaction was started at 37° C. The results showed thatincreasing the pre-incubation time up to 3 hours did not result infurther enhancement of the inhibition of haemolysis. This means that thekinetics of C6 binding by 7E5 in this reaction are such that the C6 iseffectively complexed and neutralized completely within minutes.

Example 3: Epitope Mapping of 7E5 Monoclonal Antibody

Peptide arrays were used to determine the epitope of 7E5 in human C6.Consecutive overlapping 16 mer peptides (peptides 16 amino acids long,overlapping 14 amino acids) from the C6 protein sequence weresynthesized and spotted in a grid pattern on a membrane. The membranewas then incubated with 7E5 antibody to detect which peptide wasrecognized by the antibody. The primary peptide sequence recognized by7E5 was GSCQDGRQLEWGLERT (peptide 418) (SEQ ID NO: 1).

Subsequently, an alanine scan (in which alanine is used to replace aminoacids one by one) on selected peptides was performed to help pinpointthe epitope. In this study, in addition to modifications of peptide 418,a peptide with 4 amino acids shifted relative to 418, peptide 420(DGRQLEWGLERTRLSS) (SEQ ID NO: 2), and several of its alaninemodifications, showed binding of 7E5. Thus, it was concluded that aminoacids which form a portion of the main epitope of 7E5 are expected to bewithin this peptide sequence combining peptides 418 and 420:GSCQDGRQLEWGLERTRLSS (SEQ ID NO: 3).

FIG. 4A shows the sequence of peptide 418 and surrounding area in human(SEQ ID NO: 50) and rat C6 (SEQ ID NO: 51). As illustrated schematicallyin FIG. 4B, peptide 418 is partially located at the end of the first FIMdomain of C6.

To determine whether other antibodies bind to the same epitope as the7E5 antibody, an Biacore cross blocking experiment was conducted inwhich the C6 antigen was coupled to the chip, followed by flow of theanalyte(s), which was either a single anti-C6 antibody alone (as acontrol) or a first anti-C6 antibody (Antibody 1) followed by a secondanti-C6 antibody (Antibody 2) to determine cross-blocking. The resultsfor the cross-blocking experiment to determine whether the mouse mAb27B1 binds the same epitope as the rat mAb 7E5 are shown in FIG. 3A-D,wherein FIG. 3A shows the results with 27B1 as Antibody 1 and 7E5 asAntibody 2, FIG. 3B shows the results with 7E5 as Antibody 1 and 27B1 asAntibody 2, FIG. 3C shows the results for 27B1 alone and FIG. 3D showsthe results for 7E5 alone.

Example 4: In Vivo Efficacy of 7E5 Monoclonal Antibody

To test whether 7E5 is able to block C6 in a living animal, C6 deficientPGR rats supplemented with human C6 were used. This approach was usedbecause 7E5 is specific for human C6 and is unable to block rat C6. Inthe C6 deficient rats, human C6 can be injected to restore fullcomplement system functionality and MAC activity, and the effect of 7E5can be measured without confounding effects caused by rat C6.

First, this approach was tested by determining the haemolytic activityin two rats injected with human C6. By taking several blood samples intime after injection of C6, the half-life of human C6 in the rats wasestimated to be about 48 hours. Two C6 deficient rats were injected IVwith 4 mg/kg of human C6. Blood samples were drawn 10 minutes, 24 hoursand 48 hours post injection of C6. After coagulation of all bloodsamples, serum was isolated by spinning down the coagulate (13,000 rpmin an Eppendorf table top centrifuge for 10 minutes at roomtemperature). The serum was used in the haemolytic assay described inExample 1 to determine MAC activity. Serum from a wild type PVG rat anda non-treated C6 deficient rat were used as references for maximal andminimal haemolytic activity. Using the haemolytic assay, the half-lifeof human C6 in the rats was estimated to be about 48 hrs.

In a pilot experiment, one female C6 deficient PVG rat (220 grams bodyweight) was injected with a high 12 mg dose of 7E5 intraperitoneally andsupplemented with 2 mg of human C6 (intravenous injection). Human C6 wasisolated from human serum using affinity purification with C6 antibodycoated columns. C6 was dosed 1 mg 24 hours before and 1 mg 5 minutesafter 7E5 bolus injection. The control rat (same weight as the 7E5treated rat) received only the C6 injection. Blood for the haemolyticassay was drawn 60 minutes after injection of 7E5. The results are shownin FIG. 5. The results showed that haemolytic activity was blocked by7E5 60 minutes after 7E5 dosing thus proving that 7E5 can block MACformation in vivo.

In a subsequent experiment, two C6 deficient female rats (PVG strain)were injected with 1 mg C6. Blood samples were taken before C6 injectionand after C6 injection (IV 1 mg) to establish normal and supplementedhaemolytic activity. 10 minutes after C6 injection, 7E5 was dosed ateither 8 mg IP or 2 mg IV. 60 minutes after 7E5 dosing, blood sampleswere taken to assess the effect of 7E5 on haemolytic activity. Bothdosing strategies blocked MAC activity in the blood. Then another 1 mgof C6 was injected IV in the same rats. Blood sampling in the 15 minutesafter the new C6 supplementation showed only modest increase inhaemolytic activity inferring that haemolytic activity remainedinhibited in both rats by free circulating 7E5.

These above-described experiments demonstrate that 7E5 is able to blockC6 in a living animal.

Example 5: Sequencing and Recombinant Expression of 7E5 MonoclonalAntibody

The nucleotide and amino acid sequences of the heavy and light chainvariable regions of the 7E5 mAb were determined by standard procedures.

The nucleotide sequence of the VH region is as follows:

(SEQ ID NO: 4) gaggtgcagctggtggagtctgatggaggcttagtgcagcctggagggtccctgaaactctcctgtgtagcctcaggattctctttcagtgactattacatggcctgggtccgccagggtccaacgaaggggctggagtgggtcgcaaccattaattatgatggtagtagtacttactatcgagagtccgtgaagggccgattcactatctccagagataatgcgaaacgcaccctatacctgcaaatggacagtctgaggtctgaggacacggccacttattactgttcaagaccttctacggaggccctgtttgcttactggggccacggcactctggtca ctgtctcctca 

The amino acid sequence of the VH region is as follows:

(SEQ ID NO: 5) EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSR PSTEALFAYWGHGTLVTVSS

The amino acid sequences of the VH CDR1, CDR and CDR3 are as follows:

CDR1: (SEQ ID NO: 6) DYYMA CDR2: (SEQ ID NO: 7) TINYDGSSTYYRESVKG CDR3:(SEQ ID NO: 8) PSTEALFAY

The nucleotide sequence of the VL region is as follows:

(SEQ ID NO: 9) gatgttgtgctgacccagactccatccacattatcggctaccattggacaatcggtctccatctcttgcaggtcaagtcagagtctcttaaatgatgttggaaacacctatttatattggtatctacagaggcctggccaatctccacagcttctaatttatttggtctccgacctgggatctggggtccccaacaggttcagtggcagtgggtcaggaacagatttcacactcaaaatcagtggagtggaggctgaggatttgggaatttattactgcatgcaagctagtcatgctccgtacacgtttggagctgggaccaacctggaactgaaa

The amino acid sequence of the VL region is as follows:

(SEQ ID NO: 10) DVVLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAP YTFGAGTNLELK

The amino acid sequences of the VL CDR1, CDR and CDR3 are as follows:

CDR1: (SEQ ID NO: 11) RSSQSLLNDVGNTYLY CDR2: (SEQ ID NO: 12) LVSDLGSCDR3: (SEQ ID NO: 13) MQASHAPYT

Following introduction of appropriate restriction sites for cloning andoptimization of the coding sequence for expression in the productioncell line (Hek-293 cells), expression cassettes were prepared. Thesynthesized heavy and light chain variable domains of 7E5 were clonedinto the pMQR eukaryotic expression vector set (pMQR-hIgG1 andpMQR-hIgK), thus generating a human-rat chimeric recombinant antibody.Sequence analysis of the resulting clones indicated that both sequenceswere cloned correctly. The pMQR eukaryotic expression vectors harboringboth 7E5 variable domains were transfected into Hek-293 cells and thesecells were allowed to produce the recombinant antibody. Followingproduction, hIgG1/hIgK antibodies were detected in the spent supernatantby means of capture ELISA. The transfection supernatant was shown tocontain recombinant 7E5 at 0.019 mg/ml.

Example 6: Humanization of 7E5 Monoclonal Antibody

As an alternative to antibody humanization methods based on cycles ofsite-directed mutagenesis, the rat 7E5 mAb was humanized using ahumanization approach based on CDR-homology between human and murineantibodies as described by Hwang and colleagues (Methods. 2005.36:35-42). This method is based on the principle that if a non-human anda human antibody have similarly structured CDRs, the human frameworkswill also support the non-human CDRs, with good retention of affinity.In this method, the human framework sequences are chosen from the set ofhuman germline genes based on the structural similarity of the humanCDRs to those of the antibody to be humanized (same Chothia canonicalstructures). A phage display library of Fab variant sequences,containing deviating FR residues, is generated. After affinity-drivenselections, individual clones are screened for binding and off-rate andthe sequence human identity and homology is determined.

The process to humanize 7E5 rat antibody applied in this work consistedof the following steps:

1—Design of humanization library: Identification of the closest humangermlines and identification of the rat VH and VK FR residues deviatingfrom these human germlines.

2—Assembly of the 7E5 gene libraries (using overlapping oligonucleotidesto synthetically generate the variable heavy (VH) and light (VL) chainencoding genes via PCR).

3—Cloning of these gene libraries into a phagemid (pCB13-CK1/3)containing the human constant heavy (CH1) and light (Cκ) chain (libraryconstruction).

4—Selection of the functional Fabs using phage display and affinityselection.

5—Screening for off-rate (Biacore) and sequencing.

6—Selection of the Fabs with the highest human identity and homologywithout loss of binding to hC6.

7—Production and purification of eight humanized leads to be used infurther affinity measurements and in functional assays.

Design of Humanization Library:

Using the nucleotide and amino acid sequences of the variable domains ofthe rat 7E5 antibody and public databases ant tools, it was confirmedthat 7E5 uses IGHV5S45*01, IGHD1-6*01, IGHJ3*01 and IGKV2S27*01,IGKJ2-3*01 as germline segments. It was also concluded that thecanonical fold combinations for CDR H1 and CDR H2 of 7E5 is 1-3 and forCDR L1 and CDR L2 of 7E5 is 4-1.

Comparison of the 7E5 VH sequence with human germlines with theidentical canonical fold combination 1-3 for CDR1 and CDR2 revealedhuman germline VH3 family member 1 as the closest match. The closesthuman JH germline is IGHJ4. The alignment against these germlinesegments is shown in FIG. 6A. The 7E5 heavy chain amino acid sequence isalso shown in SEQ ID NO: 5. The human germline VH3_1 amino acid sequenceis also shown in SEQ ID NO: 48. The FRs and CDRs are indicated, whichenables the identification of FR residues deviating from the humangermlines.

Using a similar analysis it was determined that for the 7E5 Vκ sequencethe closest human germline is human VK2 family member 5. The closesthuman JH germline are IGKJ2 and IGKJ5. The alignment against thesegermline segments is shown in FIG. 6B. The 7E5 light chain amino acidsequence is also shown in SEQ ID NO: 10. The human germline VK2_5 aminoacid sequence is also shown in SEQ ID NO: 49. The FRs and CDRs areindicated, which enables the identification of FR residues deviatingfrom the human germlines.

As shown in FIGS. 6A and 6B there were 13 positions for 7E5 VH sequencesand 16 for 7E5 Vκ, respectively, for which the human residues wereincorporated for the humanization libraries but also the rat residue incase the change would be detrimental for antigen binding. Taking intoaccount the number of positions to mutate and the number of variants perposition, the library size to cover the introduced diversity would be8.2×10³ and 9.8×10⁴ for humanized VH and Vκ libraries respectively.

Humanized 7E5 Fab Library Construction:

For the construction of the final humanized 7E5 Fab phage displaylibrary, initially two different sub-libraries were constructed:

1—VH Humanized Fab sub-library, in which the humanized 7E5 VH gene wascloned together with the WT 7E5Vκ into pCB13-CK3 phagemid, containingthe genes coding for the human constant domains CH1 and Cκ.

2—VL Humanized Fab sub-library, in which the humanized 7E5 Vκ gene wascloned together with the WT 7E5VH into phagemid vectorspCB 13-CK1 andpCB13-CK3, containing the genes coding for the human constant domainsCH1 and Cκ.

Due to the cloning strategy and sequences of the two different phagemidsused, the residues in positions 104 to 107 of the Light chain V domainof clones produced from pCB13-CK1 would correspond to LEIK (Humanized7E5 sequence), while V domain light chains of clones produced inpCB13-CK3 would show in the same positions amino acids LELK (7E5 WT Vκsequence).

The two resulting sub-libraries were panned against human C6 and bindingclones were recovered to proceed to final Fab library construction inwhich both heavy and light chain were humanized.

Synthetic Gene Assembly:

To construct the different humanized heavy and light chainsub-libraries, the humanized 7E5 VH and Vκ genes were generated by geneassembly (Cherry, J. et al. (2008) J Biochem Biophys Methods, 70:820-2;Stemmer, W. P. et al. (1995) Gene, 164:49-53).

Humanized 7E5 VH and Vκ Sub-Libraries Construction:

For the construction of the 7E5 VH Fab sub-library, the synthetic VHgenes of approximately 400 bp generated by gene assembly and a DNAfragment codifying for 7E5 Vκ WT were cloned into the phagemid pCB13-CK3(containing the human constant heavy and kappa light chain encodinggenes).

For the construction of the 7E5 Vκ Fab sub-library, the synthetic Vκgenes of approximately 400 bp generated by gene assembly and a DNAfragment codifying for 7E5 VH WT were cloned via ApaLI/XhoI sites andNcoI/NheI respectively into an equimolar mixture of phagemids pCB13-CK1and pCB13-CK3 (containing the human constant heavy and kappa light chainencoding genes).

The new vectors resulting from the cloning process were transformed byelectroporation into E. coli TG1 cells. The size of the libraries wascalculated from 5 μl spots of TG1 transformed cells on LBA Carbenicillin(100 μg/ml), Glucose 2% and the percentage of Fab inserts was determinedby colony PCR. The size and insert percentage of the sub-libraries issummarized below in Table 5.

TABLE 5 Size and Insert Percentage Obtained for Humanized 7E5Sub-Libraries Diversity Coverage Final Maximal (excess over LibraryTheoretical theoretical Sub-Library Library Size Insert % Size Diversitydiversity) Humanized 7E5 VH 2.4 × 10⁸ 95% 2.3 × 10⁸ 8.2 × 10³ ~28,000fold Humanized 7E5 Vκ 1.7 × 10⁸ 91% 1.5 × 10⁸ 9.8 × 10⁴ ~1,500 fold

Sub-libraries were also QCed by DNA sequence analysis of 48 clones perlibrary. Amino acid sequences were extracted using CLC Main WorkbenchSoftware. Analysis of valid VH and Vi sequences and of the frequency ofWT or mutated residue per position revealed that the sub-libraries weresuccessfully designed and constructed with the ratio of WT/mutation ofapproximately 50/50 (33/33/33 for position 103 in the Vκ gene) and theaverage number of FR mutations was obtained as designed.

Panning Selections of Humanized 7E5 VH and Vκ Sub-Libraries:

Phage were prepared from the two sub-libraries and used for a firstround selection on coated human C6. The aim of this round of selectionwas to clean up the sub-libraries from non-binding Fabs and therefore nostringent conditions were applied.

For the panning selections 5 and 0 μg/ml of human C6 were coated in96-well Maxisorp plate (Nunc) and blocked with low-fat milk powder(Marvell 4% in PBS). After 2 hours of incubation with sub-library phageand subsequent washes, trypsin elution (10 mg/ml) was performed at roomtemperature. Protease activity was immediately neutralized by applying16 mM protease inhibitor ABSF.

All phage outputs were infected into logarithmically grown E. coli TG1cells and 5 μl of the infected bacteria were plated on agar plates(LBAGluc 2% Carb 100 g/ml) for analysis of outputs and for enrichmentdetermination. Enrichment was calculated as the ratio between the numberof phage eluted from human C6 versus those eluted from the no proteinconditions. Very good enrichments compared to background (PBS) for bothHumanized 7E5 Vκ and VH Fab sub-libraries were observed.

Construction of Final Humanized 7E5 Fab Phage Display Library:

The final humanized 7E5 Fab library was constructed by combining therecovered humanized heavy chains (VHCH) from clones selected from the7E5 VH Fab sub-library with the recovered humanized light chains (VκCκ)selected from the 7E5 Vκ Fab sub-library. The size of the resultinglibrary was calculated from 5 μl spots of TG1 transformed cells on LBACarbenicillin (100 g/ml), Glucose 2% and the percentage of Fab insertswas determined by colony PCR.

Selections of Humanized 7E5 Fab Library:

In order to select humanized variants with no loss of affinity, or evenwith improved affinities, when compared to the rat WT 7E5 antibody,in-solution phage display selections with the humanized 7E5 Fab librarywere performed using biotinylated hC6 antigen. Human C6 was biotinylatedand QCed by SDS-PAGE, Western Blot and ELISA using the anti-human C6antibody 7E5 to detect the biotinylated C6 captured on neutravidincoated plates. Three consecutive rounds of affinity driven selectionswere performed in which the antigen concentration was decreased fromround to round, as well as the phage input was also decreased from round1 to round 2. In the second and third round of selections, phageincubated with neutravidin-captured human C6 were also incubated in thepresence of an excess of non-biotinylated C6 for 2 hours or overnight(off-rate selections) in an attempt to, after several washings, get ridof high off-rate binding clones. As control, in parallel, similarselections were performed where the phage were incubated withneutravidin-captured human C6 and PBS instead of non-biotinylated hC6(no off-rate selections).

All phage selection outputs were infected into logarithmically grown E.coli TG1 cells and 5 μl of the infected bacteria were plated on agarplates (LBAGluc 2% Carb 100 μg/ml) for analysis of outputs and forenrichment determination. Enrichment was calculated as the ratio betweenthe number of phage eluted from human C6 versus those eluted from the noprotein conditions. Very good enrichments compared to background (PBS)were obtained.

Binding Screening of Clones Selected from Humanized 7E5 Fab Library:Individual colonies of E. coli TG1 infected with the eluted phage poolsobtained after the second and third round of off-rate selections weregrown at 37° C. for 8 hours in two 96 well plates (Master plates)containing 100 μl of 2TYGlucose 2% Carbenicillin 100 μg/ml, stored in20% glycerol at −80° C. and used for later sequencing, and periplasmicextract production. A total of two master plates (MPs) were generatedwith clones from the second round selections and from the third roundselections. From these MPs, bacterial extracts containing solublemonoclonal Fabs (periplasmic extracts) were produced. Monoclonalbacterial small-scale cultures were induced at OD₆₀₀ of 0.8 by addingisopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM. The periplasmic extracts (P.E.s) containing Fabs were then preparedby freezing-thawing of the bacterial pellet in PBS and subsequentcentrifugation to remove cell debris.

In order to determine the target binding capacity of the selectedclones, P.E.s at 1:5 dilution were tested for binding to 10 nM ofbiotinylated hC6 captured on neutravidin-coated Maxisorp plate. P.E.prepared from rat 7E5 WT Fab was used as positive control. Blank P.E.(prepared from non-inoculated well in the MP) was used as negativecontrols. Binding of P.E.s to the target was detected with an anti-c-mycmouse antibody conjugated to Horseradish peroxidase (HRP). A binding hitrate of 40% was obtained for both MPs and binding signals (O.D. 450 nmvalues) of the positive clones were comparable to the signal obtainedwith the parental rat 7E5 Fab.

Off-Rate Screening and Analysis of Human Identity and Homology ofSelected Clones Binding Human C6:

For the positive binding clones, the off-rate for hC6 was determinedusing the SPR method and in parallel, the DNA coding for the variabledomain of the heavy and of the light chains was sequenced.

To determine the off-rate a Biacore 3000 (GE Healthcare) was used. Forthat purpose, 50 μg/ml of hC6 in acetate buffer pH 4.5 was immobilizedon a CM5 sensor chip (GE Healthcare BR-1000-12) to approximately 2000RU. Regeneration conditions were tested and 2×10 μl of 10 mM NaOH and 1M NaCl were used for the regeneration between sample injections. 30 μlof P.E.s, prepared as described above, were diluted in 120 μl of HBS-EPbuffer and from this 60 μl were injected with a flow of 30 μl/min.Dissociation was measured during 400 seconds and the off-rate wasdetermined by applying the 1:1 Langmuir dissociation fitting model.

In parallel, to analyze human identity and homology the DNA coding forthe variable heavy chain and light chain of clones that showed specificbinding to hC6 was sequenced.

Amino acid sequences were extracted using CLC Main Workbench Software.The Vκ and the VH sequences were aligned separately against thereference sequence (7E5 WT). All sequences were analyzed to determinethe percentage of human identity (fraction of framework residues whichis found in the closest matching germline) and human homology (fractionof framework residues which is found in the closest matching germline orother germlines of the same subclass) using the Abligner software.

Overall a good correlation was observed between the ELISA and theBiacore data, and also good human identity and homology percentagevalues varying from 88-99%.

A lead panel of eight clones that had good binding, off-rate and humanidentity and homology data were selected, referred to as 8G09, 7E12,7G09, 8F07, 7F06, 7F11, 7E11 and 7F02. The complete nucleotide and aminoacid sequences of the variable domains of the heavy and light chains ofthe lead panel of eight humanized clones are shown below:

8G09 VH and VL Nucleotide Sequences:

8G09 VH (SEQ ID NO: 14)GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAACGCACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTGTCTC CTCA 8G09 Vκ(SEQ ID NO: 15) GATATTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGATCAAA

7E12 VH and VL Nucleotide Sequences:

7E12 VH (SEQ ID NO: 16)GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGGCTGAGGACACGGCCACTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTC CTCA 7E12 Vκ(SEQ ID NO: 17) GATGTTGTGCTGACCCAGACTCCATCGACATTATCGGTTACCCCTGGACAACCGGCCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAATTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGACAGGGGACCAACCTCGAGATCAAA

7G09 VH and VL Nucleotide Sequences:

7G09 VH (SEQ ID NO: 18)GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAACGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTC CTCA 7G09 Vκ(SEQ ID NO: 19) GATGTTGTGCTGACCCAGACTCCATCGTCATTATCGGTTACCCCTGGACAATCGGCCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAATTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGACAGGGGACCAAACTCGAGCTGAAA

8F07 VH and VL Nucleotide Sequences:

8F07 VH (SEQ ID NO: 20)GAGGTGTAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCAGGATTCTCTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTC CTCA 8F07 Vκ(SEQ ID NO: 21) GATGTTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTGGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAAACTCGAGATCAAA

7F06 VH and VL Nucleotide Sequences:

7F06 VH (SEQ ID NO: 22)GAGGTGTAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGGGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACAGCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTC CTCA 7F06 Vκ(SEQ ID NO: 23) GATGTTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAACCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGCTGAAA

7F11 VH and VL Nucleotide Sequences:

7F11 VH (SEQ ID NO: 24)GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAACGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTTCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCT CCTCA 7F11 Vκ(SEQ ID NO: 25) GATGTTGTGCTGACCCAGACTCCATCGACATTATCGGTTACCCCTGGACAACCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTGGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGATCAAA

7E11 VH and VL Nucleotide Sequences:

7E11 VH (SEQ ID NO: 26)GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTGTCTC CTCA 7E11 Vκ(SEQ ID NO: 27) GATATTGTGCTGACCCAGACTCCATTGTCATTATCGGCTACCCCTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAGGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAACCTCGAGATCAAA

7F02 VH and VL Nucleotide Sequences:

7F02 VH (SEQ ID NO: 28)GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACAGCCTATACCTGCAAATGAACAGTCTGAGGTCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCA CTGTCTCCTCA 7F02 Vκ(SEQ ID NO: 29) GATGTTGTGATGACCCAGACTCCATCGACATTATCGGCTACCCCTGGACAATCGGCCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAATTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGCTGAAA

8G09 VH and VL Amino Acid Sequences:

8G09 VH (SEQ ID NO: 30)EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPS TEALFAYWGQGTLVTVSS8G09 Vκ (SEQ ID NO: 31)DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAP YTFGAGTRLEIK

7E12 VH and VL Amino Acid Sequences:

7E12 VH (SEQ ID NO: 32)EVQLVESDGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPS TEALFAYWGHGTLVIVSS7E12 Vκ (SEQ ID NO: 33)DVVLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAP YTFGQGTNLEIK

7G09 VH and VL Amino Acid Sequences:

7G09 VH (SEQ ID NO: 34)EVQLVESDGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQGPTKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCAR PSTEALFAYWGHGTLVTVSS7G09 Vκ (SEQ ID NO: 35)DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASH APYTFGAGTRLEIK

8F07 VH and VL Amino Acid Sequences:

8F07 VH (SEQ ID NO: 36)EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPS TEALFAYWGHGTLVTVSS8F07 Vκ (SEQ ID NO: 37)DVVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAP YTFGAGTKLEIK

7F06 VH and VL Amino Acid Sequences:

7F06 VH (SEQ ID NO: 38)EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPS TEALFAYWGHGTLVTVSS7F06 Vκ (SEQ ID NO: 39)DVVLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAP YTFGAGTRLELK

7F11 VH and VL Amino Acid Sequences:

7F11 VH (SEQ ID NO: 40)EVQLVESDGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPTKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPS TEALFAYWGHGTLVTVSS7F11 Vκ (SEQ ID NO: 41)DVVLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAP YTFGAGTRLEIK

7E11 VH and VL Amino Acid Sequences:

7E11 VH (SEQ ID NO: 42)EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCAR PSTEALFAYWGQGTLVTVSS7E11 Vκ (SEQ ID NO: 43)DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYLVSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASH APYTFGAGTNLEIK

7F02 VH and VL Amino Acid Sequences:

7F02 VH (SEQ ID NO: 44)EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCAR PSTEALFAYWGHGTLVTVSS7F02 Vκ (SEQ ID NO: 45)DVVMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASH APYTFGAGTRLELK

An alignment of the amino acid sequence of the rat 7E5 heavy chainvariable region to the amino acid sequences of the heavy chain variableregions of the humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06,7F11, 7E11 and 7F02 is shown in FIG. 8A, with CDR1, 2 and 3 indicated.An alignment of the amino acid sequence of the rat 7E5 light chainvariable region to the amino acid sequences of the light chain variableregions of the humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06,7F11, 7E11 and 7F02 is shown in FIG. 8B, with CDR1, 2 and 3 indicated.The heavy chain CDR1, 2 and 3 sequences for the eight humanized variantsof the rat 7E5 antibody are the same as those in the rat 7E5 mAb (theamino acid sequences of which are shown in SEQ ID NOs: 6, 7 and 8,respectively). Likewise, the light chain CDR1, 2 and 3 sequences for theeight humanized variants of the rat 7E5 antibody are the same as thosein the rat 7E5 mAb (the amino acid sequences of which are shown in SEQID NOs: 11, 12 and 13, respectively).

Fab Expression, Purification and QC:

In order to characterize some of the 7E5 humanized variants in furtherassays (i.e., complement mediated lysis of pre-sensitized erythrocytesassay, affinity determination, melting temperatures and aggregationbehavior assays), soluble Fabs were produced and purified from the leadpanel of eight clones described above. The Fab genes of all 8 humanizedclones plus the 7E5 WT control were cloned into pCB4 expression vector(very similar to pCB13 but without the gene 3 codifying sequence) viaSfiI/NotI digestion and transformed into TG1 E. coli strain via heatshock. The sequences were confirmed using the CLC Main WorkbenchSoftware.

Production of P.E.s containing soluble Fabs from the pCB4-cloned 7E5humanized variants as well as from 7E5 WT was performed in 800 ml of2×YT supplemented with 0.1% of glucose and Carbenicillin at 100 μg/ml.After induction at OD₆₀₀ of 0.5-0.8 with IPTG to a final concentrationof 1 mM, the culture was incubated at 24° C. for at least 20 hours. Thesoluble Fabs were purified with TALON metal affinity resin.

When 500 ng of the resulting purification products were run on aSDS-PAGE several extra bands apart from the Fab specific bands (50 KDaand approximately 25 KDa under non-reduced and under reduced conditionsrespectively) were observed. To further purify these samples, a resinfrom Life Technologies that contains a VHH that specifically binds tohuman CH1 domain (CaptureSelect™ Affinity resin IgG-CH1, cat #194320005)was used according to manufacturer instructions. The concentration ofthe resulting purified protein was estimated by measuring the OD280 nmusing a micro-volume spectrophotometer and assuming a molar extinctioncoefficient on E=1.53. SDS-PAGE analysis of the purified samples showeda high level of purity. The functionality of the purified Fab wasconfirmed in ELISA where binding of serial dilutions of these Fabs to 10nM of biotinylated hC6 captured on neutravidin-coated Maxisorp plate wasexamined. All eight purified Fabs exhibited effective binding to hC6.

Biacore Analysis:

In order to determine whether humanization of 7E5 altered the bindingspecificity or activity of the resultant humanized antibodies, Biacoreaffinity analysis was performed on the eight selected humanized Fabs(7E12, 7E11, 7F2, 7F6, 7F11, 7G9, 8F7 and 8F9) as compared to the(parental) wild-type rat 7E5 mab and to the mouse 27B1 mAb. The resultsare shown in FIG. 9. The results indicate that humanization of 7E5 didnot alter the specificity or activity of the antibody.

Example 7: “Mix & Match” Characterization of Humanized Anti-C6Antibodies

In this experiment, a panel of humanized VH chains and humanized VLchains from the selected humanized anti-C6 antibodies were expressed asfull-length antibodies in mammalian cells in various combinations andwere evaluated for their functional activity.

The humanized VH chains used were the eight VH chains described inExample 6 (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02), as wellas a ninth chain, 7C02, the amino acid sequence of which is shown in SEQID NO: 46. An alignment of these nine chains is shown in FIG. 8A.

The humanized VL chains used were the eight VH chains described inExample 6 (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02), as wellas a ninth chain, 7G08, the amino acid sequence of which is shown in SEQID NO: 47. An alignment of these nine chains is shown in FIG. 8B.

The heavy and light chain nucleotide sequences were cloned intoexpression vectors to create coding sequences for full-length chainshaving a stabilized IgG4 (S228P) constant region. The 9 heavy chains and9 light chains were co-expressed as pairs in every possible combinationin CHO host cells. Thus, all 81 possible “mix and match” combinations ofthe 9 heavy chains and 9 light chains were evaluated. The 81 pairs wereeach tested in the hemolytic assay and in the MAC ELISA. For each assay,4 μg of humanized 7E5 mAb from CHO supernatant was used. The results forthe hemolytic assay are shown in FIG. 7A. The results for the MAC ELISAare shown in FIG. 7B. The results demonstrate that all 81 possible “mixand match” combinations of the 9 VH and 9VL chains exhibited stronginhibitory activity in both assays.

Example 8: Animal Model for Testing Effect of C6 Antibodies on NerveRegeneration

The nerve crush model (crush of the nervus ischiadicus) is used to testthe effect of anti-human C6 monoclonal antibodies, such as rat 7E5 orhumanized 7E5, on the recovery of sensory function in C6-knock-out rats(PVC) supplemented with human C6. The nerve crush is a model forperipheral nerve injury. See WO 2010/005310 (PCT/NL2009/050418); and deJonge et al (2004) Hum Mol Genet. β (3):295-302.

For treatment, C6−/− Rats (PVG, 6-8 weeks) were supplemented with eitherhuman C6 or control (PBS). C6 was administered intravenously in C6^(−/−)rats at a dose of 4 mg/kg in PBS one day before the crush injury (day−1) and once a day on days 0-6. The C6-supplemented rats and the controlgroup were treated with anti-human C6 mAb starting 10 minutes beforecrush (day 0) (4 mg/rat intra peritoneal injection). PVG rats weretreated again with anti-human C6 mAb (4 mg/rat IP) 5 minutes before thenerve crush. Subsequent dosages of anti-human C6 mAb were given on days1-6 (4 mg/rat IP). Control animals received the same nerve crush butwere not treated with antibody. A subset of animals was sacrificed at 72hours post crush to study histology of nerve. 72 hours was chosenbecause Wallerian degeneration is then maximal in WT animals and thistime point is very informative for assessing treatment efficacy.

The nerve crush was performed as follows. All the surgical procedureswere performed aseptically under deep isoflurane anesthesia (2.5 vol %isoflurane, 1 L/min 02 and 1 L/min N20). The left thigh was shaved andthe sciatic nerve was exposed via an incision in the upper thigh. Thenerve was crushed for three 10 second periods at the level of thesciatic notch using smooth, curved forceps (No. 7), resulting in acompletely translucent appearance of the crushed area on the nerve. Theright leg was used as internal control. The muscle and the skin werethen closed with sutures.

Shown below in Table 6 is the experimental set up for treatment with therecombinant anti-human C6 mAb 7E5 (12 mg/kg):

TABLE 6 Experimental Set Up for Nerve Crush Experiment ratReconstitution pre- Treatment Crush Post- number Temgesic (4 mg/kg)bleed (12 mg/Kg) injury bleed 1 yes C6 Yes 7 E 5 yes yes 2 yes C6 Yes 7E 5 yes yes 3 yes C6 Yes 7 E 5 yes yes 4 yes C6 Yes 7 E 5 yes yes 5 yesC6 Yes 7 E 5 yes yes 6 yes C6 Yes PBS yes yes 7 yes C6 Yes PBS yes yes 8yes C6 Yes PBS yes yes 9 yes none Yes PBS yes yes 10 yes none Yes PBSyes yes

At 3 days post-injury, all the animals were intracardially perfused with4% paraformaldehyde in piperazine-N—N′-bis(2-ethane sulfonic acid)(PIPES) buffer, pH 7.6. Left and right sciatic nerves were removed fromeach animal, and one segment of 5 mm length was collected distally fromthe crush site. Each segment was conventionally processed into paraffinwax for immunohistochemistry.

Seven micron thick paraffin sections were mounted on Superfrost Plusglass slides (Knittel Glass, Germany). Sections were deparaffinated andrehydrated. Epitopes were exposed by heat-induced antigen retrieval in10 mM sodium citrate buffer (pH6.0). Non-specific binding of antibodieswas blocked using 10% normal goat serum (DAKO, Glostrup, Denmark) in PBSfor 20 minutes at room temperature. Primary antibodies were diluted inNormal Antibody Diluent (Immunologic, Duiven, the Netherlands) andincubated for 1 hour at room temperature. Detection was performed byincubating the sections in either goat anti-rabbit fluoresceinisothiocyanate (FITC)-conjugated or sheep anti-mouse Cy3-conjugated IgGfrom Sigma-Aldrich (St. Louis, Mo.) diluted 1:200 in 1% bovine serumalbumin. When indicated, slides were counterstained with4,6-diaminodine-2-phenylindole (DAPI) (Sigma-Aldrich) and mounted withVectashield mounting medium (Vector Laboratories, Burlingame, Calif.).Images were captured with a digital camera (DP 12; Olympus, Zoeterwoude,The Netherlands) attached to a fluorescent microscope (Vanox, AHBT3;Olympus, The Netherlands).

The results are shown in FIG. 10. Cells were stained with anti-C9, fordetecting MAC, anti-pan-neurofilament (SMI312) for detecting axons,anti-myelin basic protein (MBP) for detecting myelin and anti-lysosomalmembrane (CD68) for detecting phagocytic cells (macrophages). Panel Ashows the results for the uninjured sciatic nerve, showing absence ofMAC, strong axonal staining, annular myelin staining and no activatedmacrophages. Panel B shows the results after injury, in which a rat withnormal complement activity showed MAC deposition, loss of axons andmyelin and influx of macrophages. Panel C shows the results aftertreatment of the C6-reconstituted rat with anti-C6, demonstrating thatthe antibody completely blocks MAC formation, inhibits axon and myelindestruction and inhibits macrophage influx. Panel D shows the resultsfor the C6−/− rats that were not reconstituted, showing an absence ofMAC deposition and rapid nerve degeneration.

Thus, the results for the nerve crush experiment demonstrate that invivo treatment with the 7E5 anti-C6 antibody successfully blocked MACformation and inhibited axon and myelin destruction and reducedmacrophage influx, thereby demonstrating the effectiveness of theantibody in vivo in an animal model of peripheral nerve injury.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

SUMMARY OF SEQUENCE LISTING

SEQ ID NO: DESCRIPTION  1 GSCQDGRQLEWGLERT (Peptide 418)  2DGRQLEWGLERTRLSS (Peptide 420)  3 GSCQDGRQLEWGLERTRLSS (Peptide 418/420) 4 7E5 VH nucleotide sequence (Example 5)  57E5 VH amino acid sequence (Example 5)  67E5 VH CDR1 amino acid sequence (Example 5)  77E5 VH CDR2 amino acid sequence (Example 5)  87E5 VH CDR3 amino acid sequence (Example 5)  97E5 VL nucleotide sequence (Example 5) 107E5 VL amino acid sequence (Example 5) 117E5 VL CDR1 amino acid sequence (Example 5) 127E5 VL CDR2 amino acid sequence (Example 5) 137E5 VL CDR3 amino acid sequence (Example 5) 148G09 VH nucleotide sequence (Example 6) 158G09 VL nucleotide sequence (Example 6) 167E12 VH nucleotide sequence (Example 6) 177E12 VL nucleotide sequence (Example 6) 187G09 VH nucleotide sequence (Example 6) 197G09 VL nucleotide sequence (Example 6) 208F07 VH nucleotide sequence (Example 6) 218F07 VL nucleotide sequence (Example 6) 227F06 VH nucleotide sequence (Example 6) 237F06 VL nucleotide sequence (Example 6) 247F11 VH nucleotide sequence (Example 6) 257F11 VL nucleotide sequence (Example 6) 267E11 VH nucleotide sequence (Example 6) 277E11 VL nucleotide sequence (Example 6) 287F02 VH nucleotide sequence (Example 6) 297F02 VL nucleotide sequence (Example 6) 308G09 VH amino acid sequence (Example 6) 318G09 VL amino acid sequence (Example 6) 327E12 VH amino acid sequence (Example 6) 337E12 VL amino acid sequence (Example 6) 347G09 VH amino acid sequence (Example 6) 357G09 VL amino acid sequence (Example 6) 368F07 VH amino acid sequence (Example 6) 378F07 VL amino acid sequence (Example 6) 387F06 VH amino acid sequence (Example 6) 397F06 VL amino acid sequence (Example 6) 407F11 VH amino acid sequence (Example 6) 417F11 VL amino acid sequence (Example 6) 427E11 VH amino acid sequence (Example 6) 437E11 VL amino acid sequence (Example 6) 447F02 VH amino acid sequence (Example 6) 457F02 VL amino acid sequence (Example 6) 467CO2 VH amino acid sequence (FIG. 8A) 477G08 VL amino acid sequence (FIG. 8B) 48 Human VH3_1 sequence (FIG. 6A)49 Human VK2_5 amino acid sequence (FIG. 6B) 50Human C6 partial amino acid sequence (FIG. 4A) 51Rat C6 partial amino acid sequence (FIG. 4A) 52Human C6 full-length amino acid sequence(C6 definition in specification)

What is claimed is:
 1. An isolated antibody, which comprises heavy chainCDR1, 2 and 3 sequences shown in SEQ ID NOs: 6, 7 and 8, respectively,and comprises light chain CDR1, 2 and 3 sequences shown in SEQ ID NOs:11, 12 and 13, respectively.
 2. The isolated antibody of claim 1, whichis a humanized antibody.
 3. The isolated antibody of claim 1, whereinthe antibody comprises the heavy chain variable region sequence shown inSEQ ID NO: 32 and the light chain variable region sequence shown in SEQID NO:
 33. 4. A composition comprising the antibody of claim 1 and acarrier.
 5. A composition comprising the antibody of claim 3 and acarrier.
 6. The antibody of claim 1, wherein the antibody is of the IgG1isotype, the IgG2 isotype, or the IgG4 isotype.
 7. The composition ofclaim 4, further comprising at least one therapeutic agent selected fromthe group consisting of an anti-inflammatory agent, a disease-modifyinganti-rheumatic drug (DMARD), an immunosuppressive agents, and achemotherapeutic.